Method of protecting erythricytes, in particular for improvement of blood cytopenia

ABSTRACT

The present invention concerns a compound consisting of RNA, in particular RNA extracted from yeast, a pharmaceutical composition comprising such RNA and a method for the treatment of inflammatory and inflammatory-related disorders comprising administering to a patient in need of such treatment a pharmaceutical composition comprising an amount effective to ameliorate the symptoms of inflammation or inflammatory-related disorder of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent. The exogenous yeast RNA used in the present invention has a pronounced membrane-stabilizing action in a wide range of concentrations. At the same time, yeast RNA normalizes metabolism of arachidonic acid and levels of its key metabolites, thromboxane and leukotriene. Its anti-inflammatory action is accompanied by normalization of the activity of NO-synthetase and anti-oxidant activity.

This application is a national stage of international applicationPCT/US01/09590, which is a continuation-in-part of U.S. application Ser.No. 09/534,509 filed on Mar. 24, 2000, now U.S. Pat. No. 6,737,271 whichis hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a compound and a pharmaceuticalcomposition for the treatment of inflammation and diseases accompaniedby inflammatory processes, in particular inflammatory processes whichaffect cellular membranes. The present invention also concernstherapeutic methods to ameliorate or prevent symptoms of inflammatoryprocesses.

BACKGROUND OF THE INVENTION

Inflammatory Processes

Inflammation is generally accompanied by changes in the metabolism ofarachidonic acid, metabolism of nitric oxide, and creation of freeradicals. Anti-inflammatory non-steroid drugs (NSAIDS), such as aspirin,can block certain links of an inflammatory process, but these drugscannot stabilize damaged cellular membranes, which makes their influenceon an inflammatory process limited and insufficient.

Inflammation is a localized reaction of live tissue due to an injury,which may be caused by various endogenous and exogenous factors. Theexogenous factors include physical, chemical, and biological factors.The endogenous factors include inflammatory mediators, antigens, andantibodies. Endogenous factors often develop under the influence of anexogenous damage. An inflammatory reaction is inevitably followed by analtered structure and penetrability of the cellular membrane. At thetissue and organ level, inflammation is indicated by pain, swelling,reddening, increased temperature, and a lost function in some cases.Inflammation begins with a sub-lethal damage and terminates either witha complete recovery or long-term tissue ruination. There is no recoveryfrom an injury without an inflammation.

An immediate response to a tissue damage is realized via mediators,which are released due to the exocytosis or lysis of cells. The maininflammatory mediators are compounds of the kinine and fibrinolyticsystems, the complement system, metabolites of arachidonic acid,vasoactive amines, and other chemical compounds. The chemical mediatorsof inflammation include: histamine, serotonin, prostaglandins, CGRP,nitric oxide, among others.

An important role in inflammations is played by various reactiveoxygen-containing species. These compounds are synthesized when oxygentransforms them into very dangerous forms, producing free radicals,which are atoms and molecules with unpaired electrons. Different freeradicals have different activity levels.

The launch of an inflammation is influenced by various exogenous andendogenous agents. Endogenous factors, namely, mediators, antigens, andautogens define the nature and type of the inflammatory reaction,especially its course in the zone of injury. In the case where a tissuedamage is limited to the creation of mediators, an acute form ofinflammation develops. If immunologic reactions are also involved in theprocess, through the interaction of antigens, antibodies, andautoantigens, a long-term inflammatory process will develop. Variousexogenous agents, for example, infection, injury, radiation, alsoprovide the course of inflammatory process on a molecular level bydamaging cellular membranes which initiate biochemical reactions.

Inflammatory processes rely on the metabolism of arachidonic acid, whichconverts to prostaglandines (PG), tromboxanes (TX), and leukotrienes(LT). Prostaglandines, tromboxanes, and leukotrienes are the mainparticipants of all inflammatory processes. There are two known ways ofarachidonic acid cascade. The first way leads to the creation ofprostaglandines G₂ and H₂. This process is catalyzed byprostaglandin-cyclooxygenase. Cyclooxygenase catalyzes the production ofPGA₂, PGE₂, PGD₂, PGF_(2α), while tromboxane-synthesis with PGH₂produces tromboxane A₂ (TXA₂).

The cascade of metamorphoses of arachidonic acid, which is a product ofmembrane and phospholypase A₂, is best known. Through its cyclogenaseand lypoxygenase cascades, arachidonic acid turns into prostaglandinsand leukotrienes, respectively. The cyclooxygenase way leads to theformation of two bio-active products: prostacycline (PGI₂) andthromboxane (TXA₂). These products are involved in many inflammatoryeffects: bronchoconstriction, vazodilation, vasoconstriction, plateletaggregation, analgesia, pyrexia, et al.

Another way of arachidonic acid metabolism with 5-lipoxygenase leads tothe synthesis of leukotrienes: LTA₄, LTB₄, LTC₄, LTD₄, LTE₄, and LTF₄.These leukotrienes have a powerful anti-inflammatory andbronchoconstrictor action, and they play and important role in vascularpenetrability. Besides, leukotrienes are known as potential chemotacticfactors; they increase the migration of WBC and have a great influenceon the slow-releasing substance of anafilaxis (SRS-A).

Prostaglandines can play an important role in the development ofsystemic inflammatory reactions. In rheumatic arthritis, largequantities of PG and LT in the synovial liquid support the developmentof an inflammatory process and demineralization of bone tissuesurrounding joints. Leukotrienes are known to be the mainpatho-physiological mediators of inflammatory reactions. They influence,to a greater degree than prostaglandines, the penetrability of vesselsand the adhesion of leukocytes to vessel walls as well as thedevelopment of edema.

Prostaglandines effectively regulate the aggregation of platelets. PGE₁is a powerful inhibitor of platelets aggregation, while PGE₂, which isnormally released from platelets, stimulates this process. However, themost important role in blood coagulability is played by PGI₂, orprostacycline, which is synthesized in blood vessel walls by arachidonicacid. It is the most powerful inhibitor of platelets aggregation, whichhas vasodilator properties. Thromboxane, which is synthesized inplatelets, has an opposite action.

When endothelium is damaged, the adhesion of platelets withsubendothelium tissue and the aggregation of platelets is initiated. Themain role in this process is played by thromboxane A₂. Prostaglandin I₂,on the contrary, inhibits the aggregation of platelets. Therefore, theproportion of PGI₂ and TXA₂ is crucial for the process of coagulation.

Further, a special role in the process of recovery from inflammation isplayed by nitrogen oxide (NO). This gas easily penetrates in differentorgans and tissues and, as a free radical, has a powerful reactivity.Nitrogen oxide is a potent vasodilator, neurotransmitter, andinflammatory mediator, which plays a significant role in asthmaticinflammation.

Nitrogen oxide is produced endogenously by L-arginine amino acid andNO-synthetase. There are three known forms of NO-synthetase, two ofwhich are constituent, and one inducible. The inducible NO-synthetase,which is expressed in the epithelium cells, quickly increases itsactivity when anti-inflammatory cytokines (such as interleukin 1 beta(IL-1beta) and tumor necrosis factor (TNF-alfa) are released.

Nitrogen oxide has both positive and negative properties with respect toan inflammatory reaction. One important and potentially positiveproperty is its ability to relax the smooth bronchial muscle, whichresults in bronchodilation. Its negative properties include the abilityto help the inflammatory process by increasing chemotaxis neutrophils,monocytes, and oesinofils with the help of theguasine-monophosphate-dependent mechanism. It is believed that nitrogenoxide inhibits adhesion of leukocytes to vascular endothelium andbronchial epithelium.

NO plays an important biological role in defining basal vascular tonus,regulating contractions of myocardium, and modulating the interactionbetween thrombocytes and vascular walls (Zhou Q., Hellermann G. R.,Solomonson L. P., Nitric oxide release from resting human platelets,Thromb. Res., 1; 77(1):87–86; 1995). The role of thrombocyte activationin the pathogenesis of various thrombo-vascular conditions in humans andevidence about decreased NO-mediated effects in hypertension (Calver A.,Collier J., Moncada S., Vallance P., Effect of local intra-arterialNG-monomethyl-L-arginin in patients with hypertension: the nitric oxidedilator mechanism appears abnormal, J. Hypertens., 10(9):1025–1031;1990), diabetes (Calver A., Collier J., Valance P., Inhibition andstimulation of nitric oxide synthesis in the human foream arterial bedof patients with insulin-dependent diabet, J. Clin. Invest.,90(6):2548–2554; 1992), and artherosclerosis (Drexler H., Zeiher A. M.,Meinzer K., Just H., Correction of endotelial dysfunction in coronarymicrocirculation of hypercholesterolaemic patient by L-arginine,Lancet., 21–28; 338(8782–8783); 1546–1550; 1991) suggests that drugswhich increase the activity of NO-synthetase may effectively be used intreatment of patients. Human thrombocytes are capable of synthesizingnitric oxide. Large quantities of nitric oxide, for example, in thecells of endothelium, may be produced by intact thrombocytes, as well asby stimulated thrombocytes. Hence, nitric oxide of thrombocyte originplays an important role in the support of vascular homeostasis and otherNO-sensitive processes. (Zhou et al., 1995).

Beside some common features, inflammatory processes in each individualcase have certain distinctions related to the peculiarities offunctioning of the body organ and to the factors which caused theimpairment: i.e., viruses, microorganisms, injuries, poisoning, etc.

For example, one of the common mechanisms of heart diseases, includingacute infarct myocarditis, is a malfunction of the structure andfunction of the membrane of heart cells As a result, the synthesis ofleukotrienes, tromboxanes, etc., which have coronoconstrictor,arrythmogenic, hemoatractive and pro-aggregate action, increases(Bangham A. D., Hill M. W., Miller N., Preparation and use of liposom asmodel of biological membranes, Methods in Membrane Biology, Acad. Press,V. 1, N.Y., P. 1–68, 1974).

Another important factor in the pathogenesis of heart impairments is thecoronoconstrictor and hemoattractive (with regard to neutrofiles) actionof lipoxygenase derivatives LTC₄, LTD₄, LTB₄ (Hoshida S, Kuzuya T.,Nishida M., et al., Amer. J. Cardiol, 7; 63(10): 24E–2E; 1989; Lam B.K., Gagnon L., Austen K. F. et al., J. Biol. Chem., 15; 265(23):13438–1341; 1990; Svendsen J. N., Hansen P. R., Ali S. et al.,Cardiovasc. Res., 27(7): 1288–1294; 1993). Substances which can blockthis process can in turn reduce the size of necrosis at acute myocardialinfarction and, therefore, significantly decrease the lethality indifficult cases of heart disease, such as gross myocardial infarction.At the same time, such substances can stabilize the membranes of heartcells. In addition, it is known that coronoconstrictor andhemoattractive effects during infarct are accompanied by an increasedaggregation of platelets. Therefore, blocking this process also leads toa decrease of the size of impairment.

Further, disorders of the aggregate state of blood play an importantrole in the pathogenesis of various diseases. This is especiallyapparent in the pathogenesis of thrombo-vascular conditions in humans.It is known that a malfunction in the thrombo-vascular link ofhomeostasis is a key factor leading to disorders of the aggregate stateof blood, by causing changes. in the Theological properties of, bloodand triggering the formation of internal vascular aggregates.Thrombocyte-related injuries lead to failures in micro-circulationprocesses, which result in shortages of blood inflow to the tissue. Atthe initial stage of the formation of blood clots, platelets becomeactivated and further undergo adhesion to the injured endothelium. Lateron, they aggregate and an initial thrombocytic blood clot is formed.

Today, there is enough evidence of a close relation betweeninflammations, disorders in the aggregate state of blood, andcardiovascular conditions (Anderson J. L. Carlqist J. L., et al.,Evaluation of C-reactive protein an inflammatory marker, and infectiousserology as risk factors for coronary artery disease and myocardialinfarction, J. Am. Coll. Card., 32: 35–41; 1998).

Damages to a cellular membrane or inflammatory processes in human bodyare often accompanied by blood cytopenia. In most cases, such patientshave anemia, thrombocytopenia, or neutropenia. Anemia is accompanied bya decrease in the quantities of erythrocytes or hemoglobin, which isattributed to a blood loss, malfunction in the production oferythrocytes, increased destruction of erythrocytes, or to a combinationof these causes. In the case of thrombocytopenia, the quantity ofthrombocytes in blood is decreased, which causes a malfunction inthrombogenesis and subsequent bleeding. Neutropenia is a decrease in thecount of neutrophiles in blood, which often leads to an increasedsensitivity to various infections.

Normal formation of blood cells, or hematopoiesis, begins with ahematopoietic stem progenitor cell termed CFU-GEMM, which, in adults, isformed in the marrow and, under the influence of growth factors, istransformed in specialized blood cells. For example, erythrocytes areformed from CFU-GEMM under the influence of erythropoietin. Ifinfluenced by thrombopoietin, CFU-GEMM is transformed into thrombocytes.Similarly, under the influence of granulocyte-macrophagecolony-stimulating factor, or CFU-GEMM is transformed into granulocytesand monocytes. Also, lymphocytes originate from a lymphoid stem cell.

The most pronounced cytopenia with severe consequences occurs in cancerpatients, especially after chemotherapy and radiotherapy, in AIDSpatients and those, infected with HIV (J. Crawford, J. L. Gabrilove,Therapeutic Option for Anemia and Fatigue, Medscape, Oncology TreatmentUpdate, 2000, Medscape, Inc.).

Role of Cell Membranes in Inflammatory Processes

The functions of cell membranes and their relation to inflammatoryprocesses has been documented. It is known that the plasmatic cellularmembrane occupies a special place among the other membrane structuresand performs such important functions as barrier and transportation,provides a contact with the outside environment for the cell,participates in the regulation of cellular homeostasis, supports signalmechanisms of this regulation, and defines the cell's individuality andwholeness. The structural organization, dynamics, and functions oferythrocytal membranes and various hemolysis patterns, such as osmotic,oxide, immune (induced by hemolytic viruses, toxins, complement),detergent hemolysis, photohemolysis, etc., are well studied (see, e.g.,Bashford C. L., Alder G. M., Menestrina G., et al., Membrane damage byhemolytic viruses, toxins, complement, and other cytotoxic agents. Acommon mechanism blocked by divalent cation. J. Biol. Chem., 15;261(20): 9300–9308, 1986; Osorie e Castro V. R., Ashwood E. R., Wood S.G., Vernon L. P., Hemolysis of erythrocytes and fluorescencepolarization changes elicited by peptide toxins, aliphatic alcohols,related glycols and benzylidene derivatives, Biochim. Biophys. Acta.,16; 1029(2): 252–258; 1990).

It was demonstrated that pH variation in the outside environment upsetsthe balance of forces influencing the membrane, which leads tostructural changes and changes of the aggregation degree of membraneproteins. Two types of membrane structural changes are distinguished:those caused by pH variation in the range 7.0–6.0, and those for pHlevels below 4.5 (Zavodnik I. B., Pileckaya T. P., Acid lysis of humanerythrocytes, Biophizika., V. 42, N. 5, P. 1106–1112, 1997). In thelatter case, the membrane becomes destabilized and erythrocytal lysisfollows. It is known that at pH 4.7, pores are formed in glycocalyxerythrocytal membranes (Arvinte T., Cudd A., Schulz B., Nicolau C.,Biochim. Biophys. Acta., 19; 981(1): 61; 1989). In particular, decreasedpH levels of the environment change the confirmation, package type, andmobility of phospholipids in model membranes. Thus, aggregation ofmembrane proteins, denatured due to a decreased pH, is the reason formembrane damages and acid lysis in erythrocytes.

The pattern of erythrocytal hemolysis by HCl was proposed based on thecooperative protonation of some center located in stroma or on themembrane of erythrocyte with a subsequent creation of pores, sufficientto release hemoglobin. By studying the mechanism and pattern of the acidhemolysis process, information about the structural organization of themembrane and membrane-stabilizing actions can be obtained.

The best known endogenous stabilizers of hemolysis in erythrocytes(osmotic hemolysis is the best-studied) are albumin of blood plasma,metallic ions K+, Na+, Mg²⁺ and, especially, Ca²⁺, which modulate thecanals of plasmatic erythrocytal membranes, possibly including theproton canal (Anderson D. R., Davis J. L., Carraway K. L.,Calcium-promoted changes of the human erythrocyte membrane. Involvementof spectrin, transglutaminase, and a membrane-bound protease. J. Biol.Chem., 10; 252(19): 6617–6623, 1977), cholesterol adsorbed on thesurface of erythrocytes (Hui S. W., Stewart C. M., Carpenter M. P.,Stewart T. P. Effects of cholesterol on lipid organization in humanerythrocyte membrane, J. Cell. Biol., 85(2): 283–291; 1980), andpolyamines, which bind with the fatty-acid residues of membranephospholipids Rennert O. M., Shukla J. B., Polyamines in health anddisease Advances in Polyamine research, Raven Press, V. 2, N.Y, P.195–21, 1978). The best known activators of endogenous hemolysis inerythrocytes are long-chain fatty acids (Rybszynska M., Csordas A.,Chain length-dependent interaction of free fatty acids with theerythrocyte membrane, Life Sci., 44(9): 625–632; 1989), and especiallyfree radicals of oxygen and nitrogen (Sato Y., Kamato S., Takahashi T.et al., Mechanism of free radical-induced hemolysis of humanerythrocytes: hemolysis by water-soluble radical initiator. 18; 34(28):8940–8949; 1955; Sen T., Ghosh T. K., Chaudhuri A. K. Glucoseoxidase-induced lysis of erythrocytes. J, Exp. Biol., 33; (1): 75–76;1995; Wollny T., Yacoviello L. Propogation of bleeding time by acutehemolysis in rats: a role for nitric oxide. Am. J. Physiol. 272(6):2875–2884; 1997).

In summary, there is evidence to suggest that the structure of themembrane is altered during inflammatory processes. However, the model ofmembrane damage in the inflammatory process has not been used forscreening drugs and treating or preventing inflammation andinflammatory-related disorders.

Present Drugs Unsatisfactory

The present anti-inflammatory drugs are unsatisfactory because thedifficult and various biochemical reactions involved in inflammationsand the lack of reliable information about inflammatory pathogenesiscomplicate the experimental choice of pharmacological compounds capableto regulate inflammation. Thus, drugs are selected to have an effect onindividual components of an inflammation. So far, there is no drug ableto regulate most of the components of any inflammatory reaction.

Most of the known non-steroid anti-inflammatory drugs (NSAIDS)selectively influence certain phases of this pathological process.First, they influence the penetrability of blood vessels, which is oftenaltered in acute inflammations, and various cell reactions, which arecommon for chronic inflammations. Also, many NSAIDS influence metabolismthrough the mechanism of free radicals.

The initial screening of anti-inflammation processes typically usesthree groups of methods. First, the influence of drugs oneasily-identifiable inflammatory symptoms is studied. These includeswelling, hyperemia, necrosis, etc. A more advanced analysis includesexperimental therapy methods, using model arthritis, carditis, etc.,which are similar to human ailments. The third stage involves analysisof how the drug influences certain metabolic ways.

After the metabolism of arachidonic acid was studied in detail, manyanti-inflammatory compounds, whose action was to regulate the formationof such metabolic products, were proposed. In most cases, such drugs actas inhibitors of the metabolic enzymes of arachidonic acid. One exampleis the anti-inflammatory pharmacological combination of cyclooxygenase 2inhibitor and leukotriene A.sub.4 hydrolase inhibitor (Isakson, P. C.,Anderson G. D., Gregory, S. A., Treatment of inflammation andinflammation-related disorders with a combination of a cyclooxygenase-2inhibitor and a leukotriene A.sub.4 hydrolase inhibitor, U.S. Pat. No.5,990,148, November 1999). A similar approach was proposed on the basisof analogues of pyrimidines, a component of nucleic acids (Connor D. T.,Kostian C. R., Unangst P.C., 2-heterocyclic-5-hydroxy-1,3-pyrimidinesuseful as antiinflammatory agents, U.S. Pat. No. 5,240,929, August1993). Since these compounds are the inhibitors of key metabolicferments of arachidonic acid, 5-lipoxygenase and cyclooxygenase, theauthors suggested their use as anti-inflammatory drugs suitable fortreatment of a wide range of diseases, from allergenic conditions andrheumatoid arthritis to artheroscierosis and myocardial infarction.Other researchers recommended prostacyclin analogues for treatment ofthrombocyte aggregation and bronchoconstriction (Haslanger M. F.,Prostacyclin analogs and their use in inhibition of arachidonicacid-induced platelet aggregation and bronchoconstriction, U.S. Pat. No.4,192,891, March 1980).

However, since an inflammatory process initiates many differentmetabolic cascades, the use of inhibitors or metabolic analogues ofarachidonic acid does not allow to balance all such reactions and,hence, cannot regulate the complex inflammatory process in asatisfactory manner.

Further, aspirin, which has been used in applied medicine for a longtime, has also been proposed since it can block metabolic ferments ofarachidonic acid. Inhibitors of prostaglandines, such as aspirin, quiteeffectively influence the inflammatory processes. For this reason, theyare successfully used in clinics for the treatment of rheumatoidarthritis, osteoarthritis, and other similar inflammatory processes.Aspirin also has anti-coagulation properties, since it inhibits thesynthesis of TXA₂, and it influences at least partially the synthesis ofPGI₂. A daily dose of 3 g of aspirin is commonly used for prevention ofstenocardia, as a post-infarct and post-insult treatment, or forpatients with a high risk of cardio-vascular conditions.

However, studies on the synthesis of TXA₂ and PGI₂ in vivo have shownthat peroral administration of aspirin decreases the secretion of PGI₂only for 2–3 hours, while the secretion of thromboxane is halted for 10days (Vesterqvist O., Measurements of the in vivo synthesis ofthromboxane and prostacyclin in humans, Scand. J. Clin. Lab. Invest.48(5): 401–407; 1988). This author, as well as others (see, e.g., LorenzR. L., Boehlin B., Uedelhoven M. W., Weber P. C., Superior antiplateletaction of alternate day pulsed dosing versus split dose administrationof aspirin, Am. J. Cardiol. 15; 64(18): 1185–1188; 1989), not only showthe difficulties in administering the right dose of aspirin, but alsoprovide and experimental ground for the frequent side effects caused byaspirin during its long-term use.

Specifically, aspirin and other non-steroid anti-inflammatory drugs maybe the cause of anaphylactoid reactions in sensitive individuals. Themechanism of these reactions is dose-dependent toxic-idiosyncratic, notimmunologic. Also, aspirin is the most common cause of accidentalpoisoning. Children, treated by aspirin before poisoning, are also atgreat risk. Aspirin overdose, which occurs frequently, is difficult tocorrect. The effective aspirin dose for many diseases, includingrheumatoid arthritis, constitutes 3–6.5 mg per day, which leads toirritations of the gastro-intestinal tract. Patients withgastro-intestinal conditions do not tolerate aspirin. Aspirin alsocauses erosion, bleeding stomach ulcers, diarrhea, and duodenum ulcers.Further, aspirin is commonly used in treatment for its anti-thrombocyticaction, but it is badly tolerated and causes side-effects when taken fora long period of time. In addition, by inhibiting non-selectivelycyclooxygenesis, aspirin interferes with the synthesis of thromboxane,which is a powerful aggregant and vasoconstrictor, and may also lead todecreased levels of prostacycline, which is both anti-aggregant andvasodilator.

All these negative side-effects of aspirin and other NSAIDS motivate thesearch for new drugs which would have anti-inflammatory properties, butwhich are non-toxic in a wide range of concentration, have no sideeffects during a long-term use, and are capable of preventing andterminating inflammatory processes.

Further, a complex treatment of cytopenia is done with hematopoieticgrowth factors (J. Crawford, Hematopoietic Growth factor: CurrentPractice and Future Directions, 42-nd Annual Meeting of the AmericanSociety of Hematology, 2000, Medscape, Inc.). However, it is quitecomplicated and expensive. For example, the treatment of cytopenia inHIV-infected patients depends upon the specific cause of theabnormality, hematopoietic growth factors are used. Erythropoietin isused to treat anemia, thrombopoietin is used for thrombopenia, and G-CSFis used to treat neutropenia in HIV-infected patients. Thus, treatmentof cytopenia in HIV-infected patients requires a very expensivediagnostics of the endogenous level of such growth factors and quiteexpensive growth factors, which are commonly obtained via recombinanttechnologies.

For this reason, a search of inexpensive drugs, which could normalizeanemia, thrombocytopenia, and neutropenia is important.

Pharmaceutical Use of Nucleic Acids

Nucleic acids are commonly used in pharmacology (Rothenberg M., JonsonG., Laughlin C. et al. Oligodeoxynucleotides as anti-sense inhibitors ofgene expression: therapeutic implications, J. Natl. Cancer Inst., 18;81(20): 1539–1544; 1989; Zon G., Oligonucleotides analogues as potentialchemotherapeutic agents, Pharm. Res., 5; (9): 539–549; 1988). However,pharmaceutical uses for nucleic acids have not included inflammatory orinflammatory-related disorders. For example, Anderson et al., proposesthe method of modulating the effects of cytomegalovirus infections withthe help of an oligonucleotide, which binds with mRNA ofcytomegalovirus, for treatment of cytomegalovirus infections in humans(Anderson K., Draper K., Baker B., Oligonucleotides for modulating theeffects of cytomegalovirus infections, U.S. Pat. No. 5,442,049, Aug. 15,1995). On the basis of a specific nucleic acid, which encodes thesuccession of 3′ non-translated sector of protein kinase C, Boggs et al.propose a method for diagnosis and treatment of conditions, which areassociated with protein kinase C alpha (Boggs R. T., Dean. N. M.,Nucleic acid sequences encoding protein kinase C and antisenseinhibition of expression thereof, U.S. Pat. No. 5,681,747, October1997). Also, Yano et al. patented a DNA compound obtained fromMycobouterium bovis and Bacillus subtilis for treatment of stomachulcers (Yano O., Kitano T., Method for the treatment of digestiveulcers, U.S. Pat. No. 4,657,896, April 1987).

In particular, it is known that ribonucleic acid (RNA), products of itspartial hydrolysis, and synthetic poly-ribonucleotides have a wide rangeof bioactivity (Kordyum V. A., Kirilova V. S., Likhachova L. I.,Biological action of exogenous nucleic acids, Visnyk ASC USSR, V. 41, N.6, P. 67–78, 1977). They activate protein synthesis in cells (Sved S.C., The metabolism of exogenous ribonucleic acids injected into mice,Canad. J. Biochem., V. 43, N. 7, P. 949, 1965) and have anti-tumoractivity (Niu M. C., Effect of ribonucleic acid on mouse acids cells,Sciens., N. 131, P. 1321, 1960). RNA can increase antibody generationand decrease the inductive phase of antibody genesis (Johnson A. G.,Schmidtke I., Merrit K. et al., Enhancement of antibody formation bynucleic acids and their derivatives, in Nucleic acid in immunology,Berlin, P. 379, 1968; Merrit K., Johnson A. G., Studies on the adjuvantof bacterial endotoxins on antibody formation, 6. Enhancement ofantibody formation by nucleic acids, J. Immunol., V. 94, N. 3, P416,1965; Brown W., Nakono M., Influence of oligodeoxyribonucleotides onearly events in antibody formation, Proc. Soc. Exper. Biol. Med., 5, V.119, N. 3, P. 701, 1967). It was shown that certain increased ordecreased immunologic indicators normalize under the influence of RNA.In the first place, this applies to T-lymphocytes, cooperation of T- andB-lymphocytes, activation of macrophage function, etc.

Further, exogenous RNA is used for the DNA synthesis in dividing cellsand for the RNA synthesis in metabolizing cells. It was also determinedthat 2 hours after the introduction, exogenous RNA was included in theRNA of lymphocytes and macrophages (Enesco N. E., Fate of 14C-RNAinfected into mice, Exper. Cell Res., V. 42, N. 3, P. 640, 1966).Evidence suggests that yeast tRNA can be included into cells in the formof intact molecules (Herrera F., Adamson R. H., Gallo R. C., Uptake oftransfer ribonucleic acid by normal and leucemic cells, Proc. Nat. Acad.Sci. USA, 67(4): 1943–1950; 1970).

It was determined by analytical methods that RNA is present inpractically all membranes of animal cells (membranes of endoplasmicreticulum, mitochondrial, nucleic, and plasmatic membranes). Itscontent, depending on the type of tissue and on the method of membraneisolation, varies between 0.5 and 4% of the dry weight of the membrane.Experimental results show that special membrane RNA exists in isolatedmembranes (Shapot V. S., Davidova S. Y., Liponucleoprotein as anintegral part of animal cell membrans. Prog. Nucleic Acid Res. 11:81–101; 1971; Rodionova N. P., Shapot V. S. Ribonucleic acid of theendoplasmatic reticulum of animal cells. Biochim et Biophis Acta, 24;129(1); 206–209; 1966). The functions of membrane RNA are not fullyunderstood.

The functions of membrane RNA in ribosome have been better studied.(Cundliffe E., Intracellular distribution of ribosoms and poliribosomesin Bacillus megaterriium. J. Mol. Biol., 28; 52(3): 467–481; 1970)Ribosomal RNA is contained in bacterial membranes, in the outermembranes of nuclei, inner and outer membranes of mitochondria, innermembrane of the Goldgi apparatus, which adjoins the plasmatic membrane,in the rugged endoplasmic reticulum, in different tissues in animals,humans, plants, microorganisms, and protozoa. It is possible thatmembrane glycolipids and glycoproteins, which contain N-acetylneuraminicacid, are involved in the formation of binding sites of ribosomal RNA inribosomes, since membranes which are treated by neuronidase lose theability to bind ribosomes. (Scott-Burden T., Hawtrey A. O., Preparationof ribosome free membranes from rat liver microsomes by means of lithiumchloride. Biochem. J. 115(5): 1063–1069; 1969. Further, it is possiblethat binding sites of ribosomes and membranes are activated by thesexual hormones, and cancerogens damage this physiological mechanism.This conclusion is supported by decreased levels of membrane-bound RNAin the process of aging (Mainwaring W. J. The effect of age on proteinsynthesis in mouse liver. Biochem J. 113(5): 869–878; 1969) and aftercastration of animals (Tata J. R., The formation, distribution andfunction of ribosomes and microsomal membranes during induced amphibianmetamorphosis. Biochem J. 105(2): 783–801, 1967). Extraction of sperminefrom a membrane leads to a separation of bound RNA from the membrane(Khawaja J. A. Interaction of ribosomes and ribosomal subparticles withendoplasmic reticulum membranes in vitro: effect of spermine andmagnesium. Biochim. Biophis. Acta., 29; 254(1): 117–128); 1971). Whenmembranes are treated with RNA of native small ribosomes of myelomacells, they separate from the membranes, while large native subunits ofribosomes remain bound with the membranes (Mechler B., Vassalli P.,Membrane-bound ribosomes of myeloma cells.I.Preparation of free andmembrane-bound ribosomal fractions. Assessment of the methods andproperties of ribosomes. J. Cell. Biol. 67(1): 1–15; 1975. Also, thenucleotide components of various membrane enzymes, for example,polyA-RNA enzyme of phosphofructokinase, constitute a possible pool ofmembrane RNA (Hofer H. W., Pette D. The complex nature ofphosphofructokinase—a nucleic acid containing enzyme, Life Sci. 4(16):1591–1596; 1965).

However, nucleic acids, and in particular RNA, and compositionscontaining the same, have not been used to treat or prevent inflammatoryor inflammatory-related disorders. In particular, most of the studiesabove rely on experiments in vitro. Further, none of these methods isdirected to treating or preventing an inflammation orinflammatory-related disorder.

Need for New Drug

In view of the above, there is a need for new anti-inflammatory drugswhich would regulate disorders of the aggregate state of blood and wouldhave less negative effects than aspirin and other NSAIDS. In particular,since an inflammatory process in the initial stage is followed byalterations in the structure and functions of the membrane in the manycells involved in the inflammatory process, drugs are needed which, notonly regulate all the components of an inflammatory metabolic cascade,but also stabilize membrane structures and functions in the involvedcells. In particular, since the traditional therapy has littleeffectiveness in extensive infarcts, which are complicated by thecardiogen shock, there is a need for new drugs capable of stopping thedestruction of cardiomyocytes.

SUMMARY OF THE INVENTION

The present invention offers a compound, a pharmaceutical compositionand a method for the treatment or prevention of inflammation anddiseases accompanied by inflammatory processes. The compound is anactive ingredient consisting of RNA, in particular RNA extracted fromyeast. Yeast RNA is a heterogenous compound of low-polymeric RNA, whichcomprises various quantities of nucleotides, nucleotide polymers, andusually 5 to 25 nucleotides. Oligonucleotides and transport RNA with agreat number of minor bases prevail in yeast RNA.

Since one of the common features of all inflammatory processes at amolecular level is altered penetrability and structure of membrane, thepresent invention was made using a method of selecting drugs based ontheir ability to stabilize cellular membrane in inflammations. Thus, byanalyzing destructive mechanisms induced by various factors in plasmaticmembranes and learning about the structural elements of theirinteraction, which provide the optimal organization of a cell, it ispossible to select drugs having membrane-stabilizing action for appliedmedicine. Specifically, it has now been established that, sincemembranes contain low-molecular RNA which probably plays amembrane-stabilizing role, introduction into the body of exogenouslow-molecular RNA leads to stabilization of disturbed membranes, suchas, for example, membranes of cells involved in inflammatory processes.

Stabilization of the cell membrane by the compound of the presentinvention leads to the normalization of arachidonic acid metabolism andnitric oxide metabolism, which have a powerful anti-inflammatory actionand are the main participants of all inflammatory processes, forexample, rheumatoid arthritis, osteoarthritis, allergies (such asasthma), and other inflammatory conditions, such as pain, swelling,fever, psoriasis, inflammatory bowel disease, gastrointestinal ulcers,cardiovascular conditions, including ischemic heart disease andatherosclerosis, partial brain damage caused by stroke, skin conditions(eczema, sunburn, acne), leukotriene-mediated inflammatory diseases oflungs, kidneys, gastrointestinal tract, skin, prostatitis, andparadontosis.

The yeast RNA is effective in decreasing the activity of iNOS in thecourse of an auto-immune process, both during its initiation and in thechronic stage. This property allows the usage of yeast RNA inpathological conditions which are accompanied by iNOS induction:diabetes, tumor, hepatitis, infections, neuro-degenerate diseases(Parkinson's disease, Alzheimer's disease, multiple sclerosis,encephalitis), and others.

In addition, the use of natural molecules of nucleic acids, such as thecompound of the present invention, in large concentrations aspharmacological compounds causes no or little side effects, especiallytaking into account the fact that this compound constantly enters humanand animal bodies with food.

Further, the present invention offers a method for the treatment ofinflammation or inflammatory-related disorder comprising administeringto a mammal in need of such treatment an amount effective to amelioratethe symptoms of inflammation or inflammatory-related disorder ofribonucleic acid and a pharmaceutically acceptable vehicle, carrier, ordiluent.

Still further, the present invention offers a method of stabilizingdamaged cellular membranes which comprises administering to a mammalhaving damaged cellular membranes an amount effective to stabilize saiddamaged cellular membranes of ribonucleic acid and a pharmaceuticallyacceptable vehicle, carrier, or diluent.

Still further, the present invention offers a method of normalization ofNO-synthetase ability in a mammal which comprises administering to amammal in need of such treatment an amount effective to normalizeNO-synthetase ability in the mammal of ribonucleic acid and apharmaceutically acceptable vehicle, carrier, or diluent.

Still further, the present invention offers a method of inhibitingoxidation of components of cell membranes of a mammal, which comprisesadministering to a mammal in need of such treatment an amount effectiveto inhibit oxidation of components of cell membranes of the mammal ofribonucleic acid and a pharmaceutically acceptable vehicle, carrier, ordiluent.

Still further, the present invention offers a method of inhibitingthrombocyte aggregation, which comprises administering to a mammal inneed of such treatment an amount effective to inhibit thrombocyteaggregation of ribonucleic acid and a pharmaceutically acceptablevehicle, carrier, or diluent.

Still further, the present invention offers a method of improving alevel of at least one blood indicator which comprises administering to amammal in need of such treatment an amount effective to improve thelevel of the blood indicator of ribonucleic acid and a pharmaceuticallyacceptable vehicle, carrier, or diluent. The blood indicator is any oneof the respective levels of leukocytes, erythrocytes, thrombocytes,hemoglobin, neutrophils and hematocrites.

Still further, the present invention offers a method of preventing ortreating any one of cytopenia, anemia, thrombocytopenia, and neutropeniawhich comprises administering to a mammal in need of such treatment aneffective amount of ribonucleic acid and an acceptable vehicle, carrier,or diluent.

The ribonucleic acid may be administered in an amount within a range offrom about 0.1 mg to about 1 g per kg weight of a mammal, for examplewithin a range of from about 0.1 to about 1 g, more specifically fromabout 250 to about 350 mg.

Also, the present invention offers a compound consisting of ribonucleicacid extracted from yeast, for example a Saccharomyces cerevisiae or aCandida utilis. Preferably, the ribonucleic acid has a nitrogen contentof more then 14.5% by weight and a phosphorus content of more then 8.5%by weight, more preferably a nitrogen content of more then 14.7% byweight and a phosphorus content of more then 8.6% by weight, even morepreferably a nitrogen content of more then 15.0% by weight and aphosphorus content of more then 9.0% by weight.

Further, the present invention offers a pharmaceutical composition forthe treatment or the prevention of inflammation or inflammatory-relateddisorder, comprising ribonucleic acid and a pharmaceutically acceptablevehicle, carrier, or diluent.

DETAILED DESCRIPTION OF THE INVENTION

A complex analysis of known nucleic acids was carried out using variousin vitro and in vivo models. The models were chosen to correspond tocertain types of inflammatory processes, both of common and immunologicorigin. In the tests, the effects of ribonucleic acid (RNA), inparticular yeast RNA, was compared to the effects of existinganti-inflammatory drugs over a wide range of anti-inflammatoryactivities.

Summary of Experimental Models and Results

1. Model of Thrombocyte Aggregation In Vitro

An initial screening of exogenous nucleic acids was conducted in vitroon the model of aggregation of human thrombocytes induced by arachidonicacid (Born L. V. R The aggregation of blood platelets by difosfate andits reversal, Nature, V. 94, P. 327, 1962). Exogenous DNA and RNA fromprokaryotes and eukaryotes were analyzed. We used aspirin asrepresentative of a standard anti-inflammatory drug.

It was demonstrated that aspirin inhibited the aggregation ofthrombocytes induced by arachidonic acid to a certain level.Desoxyribonucleic acid obtained from chicken erythrocytes (DNA-CE)produced by “Reanal” (Hungary), inhibited thrombocytic aggregationwithin the range of aspirin. Further, DNA from cattle thymus (DNA-CT)produced by “Reanal” (Hungary), and transport RNA of E. coli (tRNA)produced by “Serva” (USA) inhibited aggregation of the inducedthrombocytes almost twice. The highest inhibiting effect wasdemonstrated by total yeast RNA, which dramatically inhibitedthrombocytic aggregation in a wide range of concentrations. Inhibitionof thrombocytic aggregation by yeast RNA depended on the form (acid orits sodium salt), purity, and presence of protein. RNA-F with proteinadmixtures was less effective by a third. The sodium salt of yeastRNA-PN in high concentration was only half as effective, and did not actin low concentration.

Since the model of aggregation of thrombocytes induced by arachidonicacid is recognized for the selection of anti-inflammatory drugs, theresults of this comparative test showed that nucleic acids, andespecially RNA, in particular, yeast RNA, have pronouncedanti-inflammatory properties.

2. Model of Acid Resistance of Erythrocyte Membranes In Vitro

Based on the recognition that destabilization of cellular membranes isthe main indication of an inflammatory process, we used the model ofacid resistance of erythrocyte membranes in vitro for the screening ofmembrane-protecting, and thus, anti-inflammatory properties of thedrugs. We chose rat erythrocytes to study the immune-stabilizing actionof exogenous nucleic acids. We analyzed the reactions of erythrocyticmembranes to the destructive influence of nitric oxide. We estimated themembrane-stabilizing action of exogenous nucleic acids and damagingactions of endogenous and exogenous nitrite anion by calculating theacid resistance of erythrocytes according to the kinetic method (TerskovI. A., Hittelzon I. I., Chemical (acid) erythrogram method, Biophizika,2(2): 259–266; 1957). The main idea of the method is to determinehistorical changes in the number of cells, which eventually becomehemolyzed under the influence of weak acids. The lysis of erythrocytesin acid environment undergoes three stages: penetration of hydrogen ions(protons, H⁺) through the plasmatic membrane of erythrocytes,protonation of hemoglobin and membrane proteins, and, as a result,osmotic destruction of erythrocytes.

Using this method, we estimated the influence of exogenous nucleic acidson the kinetics of the penetration of protons through the erythrocyticplasmatic membrane, which depends on the membrane's nature. The speed ofproton penetration in the cellular cytosol depends to a great extend onthe oxidation status of the lipid component (Kellogg E. W., FridovichI., Liposome oxidation and erythrocyte lysis by enzymically generatedsuperoxide and hydrogen peroxide J. Biol. Chem. 10; 252(19): 6721–6728;1977) and protein component, especially, the band 3 oxidation ofplasmatic membranes and is defined by the activity [H⁺]-ATP-ase, and theactivity of various exchangers (Sato Y., Kamo S., Takahashi T., SuzukiY., Mechanism of free radical-induced hemolysis of human erythrocytes:hemolysis by water-soluble radical initiator, Biochemistry, 18; 34(28):8940–8949; 1995; Lukacs G. L., Kapus A., Nanda A. et al, Protonconductance of the plasma membrane: properties, regulation, andfunctional role, Am. J. Physiol, 265(1 Pt 1): C3–C14; 1993).

Acid erythrograms were recorded by the kinetic method. In the in vitrotests, acid erythrograms were recorded in the presence of sodium nitrite(the damaging agent) and different concentrations of exogenous nucleicacids.

The in vitro tests, which used the oxide damage model of erythrocytes bynitrite anion, a stable metabolite of nitric oxide, demonstratedstabilizing and membrane-protector action of exogenous nucleic acids.

On the model of acid resistance of erythrocytic membranes, we tested thesame set of preparations as in the model of thrombocytic aggregation.

Yeast RNA preparations demonstrated membrane-protecting properties in awide range of concentrations. A more detailed analysis showed that themembrane-protector action of yeast RNA depends on their form (acid orsodium salt), purity, and the presence of protein. Well-purifiedribonucleic acid RNA-P, whose erythmograms in the concentrations 10 and100 μkg corresponded to the norm, showed the highest effectiveness.Sodium salt of yeast RNA-PN was less effective, especially in theconcentration 10 μkg. Protein admixtures in RNA-F resulted in a completeloss of the membrane-stabilizing action. Other preparations, tRNA,DNA-CT, and DNA-EC destabilized erythrocyte membranes at the testedconcentrations, which means that they cannot be used asanti-inflammatory drugs as advantageously despite theiranti-inflammatory properties demonstrated on other models.

3. Model of Erythrocytal Auto-Immune Reaction in Rats

We used the model of acid injury of erythrocytal plasmatic membranes tostudy the membrane-stabilizing action of exogenous nucleic acids. Aciddamages to the protein and lipid components of erythrocytal plasmaticmembranes were tested in vivo in the process of development of anauto-immune reaction (adjuvant arthritis). The biosynthesis of nitricoxide, which is an active oxidizing agent, became activated and,especially, hemoglobin of erythrocytes (Eich R. F., Li T., Lemon D. D.Mechanism of NO-induced oxidation of myoglobin and hemoglobin.Biochemistry, 4; 35(22): 6976–6983; 1966; Huot A. E., Kruszyna H.,Kruszyna R. et al., Formation of nitric oxide hemoglobin in erythrocytesco-cultured with alveolar macrophages taken from bleomycin-treated rats.Biochem.-Biophys. Res. Commun., 15; 182(1); 151–158; 1992; Kosaka H.,Harada N., Watanabe M. et al. Synergistic stimulation of nitric oxidehemoglobin production in rats by recombinant interleukin 1 and tumornecrosis factor. Biochem. Byophis. Res. Commun. 30; 189(1): 392–398;1992). Nitric oxide, as well as hydrogen peroxide, plays a crucial rolein the damage to cells, including blood cells, in the process ofdevelopment of autoimmune reactions. The anti-inflammatory cytokines(gamma-interferon, IL-1) induce expression of the inducible isoform ofNO-synthetase (iNOS).

We studied changes in the activity of NOS in rat blood in thedevelopment of autoimmune reaction (adjuvant arthritis) in order toevaluate the preparation's immune-modulating effect and to obtaininformation about possible levels of one of the most active oxidizinghemolytics, nitric oxide (in the form of its stable metabolite, nitriteanion). We calculated the activity of the enzyme NO-synthetase (NOS),which generates endogenous nitrite anion. These values characterize theprotective effect of exogenous nucleic acids against the damaginginfluence of nitrite anion on erythrocytic membranes. Our focus on thechanges in stability of erythrocytes in the process of autoimmunereactions is due to the large existing body of evidence supporting theimmune-modulating properties of erythrocytes (Karalnik B. V.,Erythrocytes, their receptors, and immunity, Uspekhi SovremennoyBiologii., V. 112, N. 1, P. 52–61, 1992; Prokopenko L. H., Siplivaya L.E., Erythrocytes as modulators of immunologic reactions, UspekhiPhiziologicheskikh Nauk., V. 23, N. 4, P. 89–106, 1992), which hasresulted in the use of the term “erythrocytal immune system”.

Development of the autoimmune process was accompanied by a substantialdecrease of acid resistance of erythrocytes during the early stage and,on the contrary, by a considerable excess over the norm during the finalstage, in comparison with the resistance of normal erythrocytes.

Yeast RNA increased membrane stability, i.e., normalized the process oftransportation of protons (which is attributed to the state of theprotein and lipid components of etrythrocytal plasmatic membranes)during the initial stage and kept it stable, close to the norm, duringthe following stages of autoimmune reaction.

Further, it was demonstrated that, during the development of anautoimmune process, activities of NOS in rat blood changed. During theinitial and final stages, an increased activation of NOS in rat bloodwas evidenced. Yeast RNA decreased NOS activity, so that at the finalstage, the activity was practically normal.

Also, development of the autoimmune process was accompanied by asubstantial decrease of acid resistance of erythrocytes during the earlystage and, on the contrary, by a considerable excess over the normduring the final stage, in comparison with the resistance of normalerythrocytes. Yeast RNA increased membrane stability during the initialstage, by normalizing the process of proton transportation, which isdependent on the state of the protein and lipid components ofetrythrocytal plasmatic membranes, and kept it stable, close to thenorm, during the following stages of autoimmune reaction.

In view of the above, the protecting activities of yeast RNA as shown onthe model of autoimmune process establish its ability to cure, not onlyallergic diseases, but other chronic inflammatory processes as well,such as arthritis, artherosclerosis, and other diseases that involveautoimmune reactions.

4. Model of Swelling Induced by Carrageenan in Rats

To screen nucleic acid's anti-inflammatory action, we used a commonmodel of inflammatory swelling of leg in mice provoked by a sub-plantarinjection of carrageenan. Carrageenan-induced swelling is sensitive tothe action of compounds which reduce capillary penetrability.

During the initial stage, a significant role in the mechanism ofanti-inflammatory effect of carragenan is played by kinine, while at thelater stage, proteolytic ferments and prostaglandins become moreimportant. The carrageenan model has a slower development and ispreserved for a sufficient time, which makes it possible to study thebiochemical mechanism of the anti-inflammatory action of a drug.Therefore, we used this model to study the influence of yeast RNA on thesynthesis of thromboxane and leukotriene. At the same time, we analyzedthe influence of yeast RNA on NO-synthetase activity.

Analysis of the anti-inflammatory action of nucleic acids in thecarrageenan model showed that they all have certain anti-inflammatoryaction. However, only yeast RNA in the concentration 10 mg of drug permouse resulted in a 50% reduction of swelling. The concentrations ofyeast RNA tested in mice represented 1 to 15 mg per mouse.Concentrations below 1 mg of yeast RNA preparation per mouse did notshow any action. In concentrations above 15 mg, reduction of swellingwas about 53–55%. Further, biochemical tests revealed a stabilizinginfluence of yeast RNA on the activity of NO-synthetase as well as onthe quantities ofthromboxane and leukotriene, which varied in the courseof swelling process.

By contrast, aspirin, which was tested at the recommended therapeuticdose of 20 mg/kg, influenced swelling to a considerably smaller extendand did not show stabilizing properties at the level of biochemicalmetabolism.

5. Model of Acute Ischemia in Rats

Further analysis of yeast RNA was conducted on the model of acuteischemia-reperfusion of myocardium in rats. This model is based on acommon fundamental mechanism in the development of a variety ofdifferent heart conditions, which includes alteration of structures andfunctions of the membranes in endotheliocytes, cardiocytes, and otherheart cells. This alteration results in the degradation of membranephospholipids and the creation of highly effective bio-active compounds,such as leukotrienes or thromboxanes, which have coronaroconstrictor,arythmogen, chemoactive, and pro-aggregant action (Bangham A. D., HillM. W., Miller N., Preparation and use of liposom as model of biologicalmembranes, Method in Membrane Biology, Acad. Press, V. 1, N.Y, P. 1–16,1974).

As the tests demonstrated, yeast RNA, injected in rats intravenously inthe concentration of 40 mg per rat, normalized heart function in acuteinfarcts. This was shown in a pronounced anti-arythmic action of thecompound and a substantial decrease of the necrosis area in ischemizedmyocardium of heart. The drug almost completely normalized NO-synthetaseactivity in blood and in the border zone of ischemized heart. Yeast RNAinjection normalized to a certain level the content of arachidonic acidin blood and heart of animals in acute infarctions. The injection ofyeast RNA almost completely normalized the levels of eukosanoids in ratblood in ischemia cases. The activity of mieloperoxidase, the markerenzyme of neutrophils which helps to evaluate the preparation'santi-oxidant action, decreased almost twice in animals with infarcttreated by yeast RNA.

The analysis of yeast RNA activity in the ischemia-reperfusion model inrats determined that the drug has a substantial stabilizing action indifferent cascades of inflammatory processes in the ischemized heart,which is expressed in its long-term anti-infarct action and a decreasedsize of the infarct area in myocardium.

On the basis of the study of yeast RNA action in ischemia-reperfusion ofanimal heart, we can conclude that yeast RNA has an anti-infarct action,or anti-inflammatory action in infarcts, through stabilization of thestructure and function of membranes in endotheliocytes, cardiocytes, andother heart cells.

6. Action of Yeast RNA on Blood Indicators

Blood samples, taken from groups of patients before and after thetreatment with yeast RNA compound, were studied by measuring quantitiesof leukocytes [WBC], erythrocytes [RBC], and thrombocytes [PLT] in 1microliter of blood, quantity of hemoglobin [HGB] in g/dl, neutrophils(NTP) and hematocrite [HCT] in percentage. Yeast RNA compound wasadministered either in capsules, in the concentration of 250 mg of yeastRNA per capsule, or in suppositories, in the concentration of 1.0 g ofyeast RNA per suppository.

Groups of patients were selected so as to study the effects of yeast RNAamong relatively healthy individuals, athletes, cancer patients, andHIV-infected individuals. The test results show that treatment withyeast RNA resulted in stabilized or improved blood indicators. Inparticular, treatment of cancer and 1HIV-infected patients with yeastRNA resulted in a stable normalization of cytopenia.

EXPERIMENTAL PROCEDURES AND TEST RESULTS Example 1 Method for ObtainingYeast RNA Example 1.1 Production of Yeast RNA

From Saccharomyces cerevisiae was obtained RNA-D and from Candida utiliswere obtained RNA-P, RNA-PN, and RNA-F. Yeast RNA extraction wasconducted with a 10–12% solution of sodium chloride at 100–110° C. TheRNA solution was separated from yeast sediment, cooled to 0° C. andacidified to pH 1–2 by hydrochloric acid. Deposited RNA was rinsed byethyl alcohol, dried and dissolved in water. The solution was brought topH 8.0–8.2 by sodium hydroxide. The solution with added pancreatin waskept at 37–40° C. for approximately 1 hour. The ferment was inactivatedby boiling; afterwards, the solution was filtrated. RNA was sedimentedby cooled ethyl alcohol, acidified by hydrochloric acid to pH 1–2, anddried. In this way, RNA-F was obtained. Further, the sediment wasfiltrated, rinsed in ethyl alcohol, and dissolved in water by addingsodium hydroxide to pH 6.2–6.5. RNA-PN was sedimented by alcohol. Thesediment was filtrated and dried. RNA-P was educed from RNA-F byadditional purification from protein by another pancreatin treatment andincubation for 1 hour at 37–40° C. Then, the ferment was inactivated byboiling for 5–10 min. The solution containing RNA-P was filtrated andsedimented by alcohol acidified to pH 1–2. The RNA-P sediment wasfiltrated, rinsed in ethyl alcohol and dried. The resulting compound hasa grey-yellowish color.

TABLE 1 Chemical Analysis of Yeast RNA Preparations Type RNA-P RNA-DRNA-F RNA-PN Nitrogen content % 15.49 15.16 14.16 14.65 Phosphoruscontent % 9.05 8.6 8.2 8.54 Biuret reaction (−) (−) (+) (−) DNA content% 1 1.1 1.2 1.1

The tested RNA (RNA-P and RNA-D) had the following properties as shownin Table 1: N≧14.7%, P(total)≧8.6%, protein (biuret reaction)—negative,DNA (colometric)—2.0%, sugars (chromatography)—negative, polysaccharidesbiological test)—negative.

Example 1.2 Absence of Toxicity

We established that yeast RNA-P and RNA-D are non-toxic. Single ormultiple doses of yeast RNA in bio-active amounts (250 to 500 mg per 1kg of body weight), taken intra-abdominally, did not lead to substantialchanges in the quantity of peripheral lymphocytes in mice. Such changeswould be a characterizing indicator for endotoxines.

Analogous results were obtained for intravenous introduction of nucleicacids. We tested variations in the quantity of peripheral leukocytes inrabbits 1–3 hours after 100 mg yeast RNA-P or RNA-D solution wasinjected intravenously. Intravenously injected solution of 0.85% NaClwas used as the standard of non-toxicity. It was demonstrated that,analogously to the standard, an injection of yeast RNA-P or RNA-D doesnot cause a variation in the number of leukocytes within 3 hours of theintroduction. In animals, which took 0.85% solution of NaCi, thequantity of leukocytes was equal to 13000±980, while those, who hadRNA-P or RNA-D, showed accordingly 12700±850 and 12900±980, which is notabnormal. When the rabbits received injections of 10 mg of proteuspolysaccharide, the quantity of leukocytes decreased in 1 hour from13050±1100 to 2900±210, and remained at that level while the test lasted(3 hours). These results prove the non-toxicity of yeast RNA. Further,when 100 mg of yeast RNA-P or RNA-D per 1 kg of body weight was given torabbits intravenously, no acute-phase C-reactive protein was determined,which indicates that there was no endotoxic action.

In addition, yeast RNA is not pyrogenic, which was shown on rabbits.Temperatures were taken 4 times a day, with 2-hour intervals, in a groupof rabbits for 2 days. On the third day, the rabbits were injected with0.85% of NaCl, and the temperatures were taken again 1, 2, and 3 hoursafter the injection. On the sixth day, the rats were divided into 3groups, two of which received intravenously 100 mg of RNA-P and RNA-D,respectively. The temperatures were taken again. The control animalsshowed temperature fluctuations within 0.1 to 0.4° C. The tested animalshad temperatures fluctuating within the same limits: 0.1 to 0.4° C.These results prove the non-pyrogenicity of yeast RNA.

Example 2 Anti-Inflammatory Action of Nucleic Acids Based on the Modelof Thrombocyte Aggregation In Vitro

We studied the anti-inflammatory action of nucleic acids on the model ofthrombocyte aggregation in vitro by the method of Born (Born L.V.R. Theaggregation of blood platelets by diphosphate and its reversal, Nature,V. 94, P. 327, 1962). Venous human blood was taken in silicon tubes ofBecton Dickson, which contained a 3.8% solution of sodium citrate. Inorder to receive thrombocytic-rich plasma, citrate blood was centrifugedat 1500 rev/mm for 7 minutes. Plasma free of thrombocytes was obtainedby centrifuging 2.0 ml of plasma taken from medium layers for 15 minutesat 3000 rev/mm. We counted the number of thrombocytes in thethrombocytic-containing plasma, which was later diluted by thethrombocyte-free plasma to the final concentration 200.0–300.0×10⁸/l.

An Aggregometer produced by “Tromlite” (Poland) was used for thrombocyteaggregation. In order to induce aggregation, arachidonic acid wasdiluted in Michaelis buffer in the proportion 1 mg/ml. Two tubes wereinserted in the aggregometer, one of which contained 0.2 ml ofthrombocyte-containing plasma, while the other one had 0.2 ml ofthrombocyte-free plasma and 0.1 ml of isotonic solution of sodiumchloride. After the device was switched on, 0.1 ml of arachidonic acidwas added to the tube containing plasma with thrombocytes. Then, thelight-transparency of thrombocyte-containing plasma was measured during5 minutes, which indicated the stage of thrombocyte aggregation.

In a variation of the test for studying the influence of nucleic acidson thrombocyte aggregation, before measuring, the solution ofthrombocyte plasma was preliminary incubated for 5 minutes at 37° C.with 0.1 ml of the nucleic acid at the corresponding concentration. 0.2ml of isotonic solution of sodium chloride was added to the tube withthrombocyte-free plasma. After incubation, the device was switched offand 0.1 ml of arachidonic acid was added to the tube with thrombocyticplasma and a nucleic acid. In 5 minutes, measuring was done to determinethe final stage of thrombocyte aggregation.

As the aggregation parameter, we used the index of aggregation of cells(IA), which is equal to:

${I\; A} = {\frac{{D1} - {D2}}{D1} \times 100\%}$

D1—optical density of thrombocyte-containing plasma with the inductionof aggregation by the arachidonic acid.

D2—optical density of thrombocyte-containing plasma, which waspreincubated with a nucleic acid and with an induction of aggregation bythe arachidonic acid.

Statistical processing of the results was done by Student criteria andwith the help of software as described in example 4.1.

The following nucleic acids, were studied: DNA-CT, DNA-EC, tRNA, andtotal yeast RNA-D in the final concentration 1×10⁻²% Aspirin in theconcentration 0.06 mg per tube, which contained thrombocytic plasma, wasalso tested as a standard anti-inflammatory agent.

The test results are shown on Table 2 below.

TABLE 2 Influence of Nucleic Acids and Aspirin on the Aggregation ofThrombocytes Induced by Arachidonic Acid RNA-D Aspirin DNA-CT DNA-ECt-RNA M 59.73 38.66 54.45 36.93 52.23 +−m 4.24 6.71 3.76 1.88 8.13 P <0.02 P < 0.2 P < 0.01 P > 0.5

The test results showed that nucleic acids in the concentration 1×10⁻²%inhibit aggregation of thrombocytes induced by arachidonic acid.Further, Yeast RNA-D in the concentration 1×10⁻²% inhibited aggregationof the induced thrombocytes almost twice as effectively as aspirin(38.66%): yeast RNA-D showed 59.73% and transport E. coli RNA had52.23%. DNA from chicken erythrocytes acted at the same level as aspirin(36.93%), while DNA from cattle thymus inhibited aggregation ofthrombocytes by 54.45%, which is almost at the level of yeast RNA. SinceDNA always contain a significant amount of RNA, it is probable that theinhibiting effect of DNA can be attributed to the RNA contained in DNA.

Further, an analysis of the influence of different concentrations ofyeast RNA on the aggregation of induced thrombocytes showed that yeastRNA was effective in a wide range of concentrations from 0.1% to 1×10⁻⁵%and inhibited aggregation by 78.5% and 14.2%, as shown in Table 3 below.

TABLE 3 Concentration-Dependence of the Influence of Yeast RNA-D on theAggregation of Thrombocytes Induced by Arachidonic Acid RNA RNA RNA RNARNA 0.1% 1 × 10⁻²% 1 × 10⁻³% 1 × 10⁻⁴% 1 × 10⁻⁵% M 78.58 53.08 28.8843.35 14.23 +−m 7.51 3.23 1.63 10.3 4.98 P < 0.01 P < 0.001 P < 0.01 P >0.001

Still further, it was demonstrated that the inhibiting effect onaggregation depends on the purity of yeast RNA and its sodium salt, asshown on Table 4 below.

TABLE 4 Influence of Yeast RNA-P, -PN and -F on the Aggregation ofThrombocytes Induced by Arachidonic Acid RNA-P RNA-PN RNA-F Conc. 0.1% M84.09 45.96 57.9 +−m 3.77 8.96 9.58 P < 0.001 P < 0.02 Conc. 1 × 10⁻²% M71.91 55.44 60.90 +−m 8.45 8.04 10.39 P < 0.2  P > 0.5  Conc. 1 × 10⁻³%M 29.76 3.72 18.26 +−m 5.36 2.4 5.46 P < 0.001 P < 0.1 

Table 4 shows that RNA-F containing protein admixtures and lower levelsof nitrogen and phosphorus content acted less effectively in the rangeof concentrations from 1×10⁻¹% to 1×10⁻³%. For example, at its highestconcentration, RNA-F inhibited thrombocytic aggregation by 57%, whereasat its lowest concentration, inhibition was only 22.7%. At the sametime, well-purified RNA-P inhibited thrombocytic aggregation by a thirdmore effectively, accordingly, by 84% and 29.7%. Also, when RNA wastransformed into its sodium salt, the anti-aggregate propertiesdecreased dramatically. Thus, RNA-PN, at its highest concentrations, wasonly half as effective (44.4%) as the acid form, while at its lowestconcentration, RNA-PN did not show any anti-aggregate properties.

Therefore, based on the model of aggregation of thrombocytes induced byarachidonic acid, it was demonstrated that RNA compounds and,especially, purified yeast RNA, have pronounced anti-aggregateproperties in a wide range of concentrations, which indicates theiranti-inflammatory action.

Example 3 Anti-Inflammatory Action of Nucleic Acid Based on the Model ofErythrocyte Membrane Stabilization In Vitro

The membrane-stabilizing and anti-radical actions of nucleic acids wereevaluated in rat erythrocytes in tests in vitro. Erythrocytal membraneswere damaged by nitrite anion, a stable metabolite of nitric oxide,which causes oxide injuries in the protein (especially, hemoglobin) andlipid components of the membrane.

In order to evaluate the membrane-stabilizing action of nucleic acidsagainst the influence of free radicals, we calculated the acidresistance of normal rat erythrocytes separated from blood plasma. Raterythrocytes were rinsed thrice in the cold (4°C.) solution of 0.15M ofNaCl. The layers of leukocytes and thrombocytes were removed. Acid lysisof the remaining erythrocytes was induced by adding 10 μl of thesuspension, which was diluted to the concentration of erythrocytes(0.7×10⁶ cells per 1 ml of iso-osmotic medium), and which contained0.14M of NaCl, 0.01M of the citrate-phosphate buffer pH=2.5, differentdoses (10 or 100 μg) of nucleic acids, and a stable concentration ofnitric sodium, 250 μg per 1 ml, to initiate the oxide damage oferythrocytes.

Erythrocytal lysis was initiated by adding 1 ml 0.004N HCI; changes inexistence were recorded at 750 nmol. The method of calculation isexplained in Example 6.3. It was demonstrated that yeast RNA-D in thedoses of 10 and 100 pg increased the level of total resistance of theerythrocytes from 288 units (control value recorded for the influence ofNaNO₂ without yeast RNA) to 449 units (yeast RNA concentration 10 μg)and 437 units (yeast RNA concentration 100 μg), which is close to norm(475 units). RNA-PN increased total resistance to 328 units in the doseof 10 μg and to 415 units in the dose of 100 μg. RNA-P increased totalresistance to 315 units in the dose of 10 μg and to 462 units in thedose of 100 μg (maximally close to the normal level of this indicator).RNA-F increased total resistance to 338 units in the dose of 10 μg and,on the contrary, somewhat decreased (to 271 units) in the dose of 100μg.

DNA-CT increased total resistance to 338 units in the dose of 10 μg andto 654 units in the dose of 50 μg (which is double the control value andeven greater than norm (without harmful influence of NaNO₂)). In thedose of 100 μg, however, its effect was theopposite—membrane-stabilizing, which was shown by a decreased totalresistance to 158 units, which is almost half the control value.

DNA-EC in the dose of 100 μg did not change acid resistance oferythrocytes in our oxide-damage model. In the dose of 10 μg, itincreased acid resistance to 408 units, which is a little lower than thecalculated protector action of RNA-D (449 units in the dose of 10 μg).

Therefore, exogenous DNA, regardless of their origin, have significantanti-stabilizing influence on cellular membranes. Since they damagecellular membranes, they cannot be used as drugs or food supplements.

The preparation of t-RNA in both doses (10 μg to 279 units and 100 μg to296 units μg) did not influence the acid resistance of erythrocytes.

The tests show that yeast RNA, when tested in vitro, showsmembrane-stabilizing and anti-radical properties which depend on itsform, origin, and purity. Well-purified yeast RNA-P, whoseanti-inflammatory properties were studied more in detail, showed thebest effectiveness.

Example 4 Anti-Inflammatory Action of Nucleic Acid Based on the Model ofLocal Inflammation Provoked by Carrageenan (LPS) Example 4.1 Action ofYeast RNA on Swelling in the Model of Local Inflammation Provoked byCarrageenan (LPS) In Vivo

To study the anti-inflammatory action of drugs, we used the model oflocal inflammation in mice. Inflammation in BALB-line mice was modeledwith the help of carrageenan, a classical phlogogenic agent. 30 minutesbefore the injection, the mice were injected intra-abdominally withdrug, which was dissolved in 2 mg of physiological solution (PS).Carrageenan (LPS) produced by Serva Fein Biochemica (Germany) wasprepared in the form of a 1% solution in PS. The obtained viscoussolution, 40 mcl, was injected subplantally in the left back leg. Theright, intact leg was taken as control. 4 hours later after thecarrageenan injection, the mice were killed via decapitation, and theirback legs were detached from the bodies on the same level, a littlehigher the ankles. After that, the legs were carefully weighted, with 1mg accuracy. Obtained results were statistically processed by theMultiFac 2.2. SPSS 8/0 software. The anti-inflammatory effect of thedrug was calculated by the formula:

${{Percentage}\mspace{14mu}{of}\mspace{14mu}{reduction}\mspace{14mu}{of}\mspace{14mu}{inflammation}}\mspace{14mu} = {\frac{V_{k} - V_{o}}{V_{k}} \times 100\%}$

-   -   V_(k)—average increase of volume (mass) of the swollen leg in        control mice    -   V_(o)—average increase of volume (mass) of the swollen leg in        treated mice

In the first test, mice were divided into 8 groups. The first groupconsisted of control animals, which were injected intra-abdominally with2 ml of PS. Also, 40 ml of PS was injected in the left leg. This groupwas studied to determine the influence of injection on the course ofinflammation in a leg. The second group, control with LPS, took 2 mg ofPS intra-abdominally and received LPS injections in the left leg. Thethird group took 2 mg of aspirin dissolved in PS in the concentration0.4 mg per mouse. In the fourth, fifth and sixth groups, yeast RNA-D wasdissolved in PS in respective concentrations 5, 10, and 15 mg in 2 ml ofPS per animal. LPS was injected in the left leg to provoke swelling. Theseventh and eighth groups were treated respectively by DNA-TC andDNA-EC, which were injected in the concentration 15 mg per mouse, asexplained above for RNA-treated groups.

The right legs were left intact. 4 hours later, the animals weredecapitated. Both legs were detached from the bodies and their masseswere studied in each group of animals. Results of these tests on theanti-inflammatory action of nucleic acids are presented in Table 5below.

TABLE 5 Influence of Nucleic Acids on Local Inflammation of Mice LegsRNA-D RNA-D RNA-D DNA-TC DNA-EC Control + PS Control + LPS Aspirin 5mg/m 10 mg/m 15 mg/m 15 mg/m 15 mg/m 0 43.31 + 35.5 + 27.4 + 28.88 +20.3 + 31.8 + 289 2.43 2.8 2.05 2.27 3.17 2.59 % in- 18.03% 36.74%47.17% 53.13% 26.58% 33.27% hibition P < 0.001 P < 0.001 P < 0.001 P <0.001 P < 0.001 P < 0.001

As shown in Table 5, aspirin in the administered concentration reducedthe development of swelling in mouse legs by 18.03%. This is consistentwith the results cited in other papers for this model and proves theadequacy of the modeled inflammation. Aspirin concentrations alsocorrespond to the dose of 20 mg/kg which is currently recommended for aclinical use and which has fewer negative consequences for a long-termuse in various forms of inflammatory processes.

Further, the preparation of yeast RNA-D showed a significantanti-inflammatory action, which directly depended on the concentration.In the concentrations 5, 10, and 15 mg per mouse, the drug inhibitedswelling by 36.74%, 47.17%, and 53.13% accordingly. The preparation ofDNA-TC and DNA-EC also showed some anti-inflammatory action, though inquite high concentrations (15 mg per mouse), and indicators of theanti-inflammatory action were twice as low (26.58% and 33.27%,respectively).

On the basis of the results, we can conclude that nucleic acids haveconsiderably improved anti-inflammatory properties as compared toaspirin, and yeast RNA has by far the most significant action.

Example 4.2 Action of Yeast RNA on Biochemical Indicia in the Model ofLocal Inflammation Provoked by Carrageenan (LPS) In Vitro

The anti-inflammatory action of yeast RNA was compared with the actionof aspirin in the dynamics of a developing inflammatory reaction(0^(th), 30^(th), 60^(th), 320^(th) min) on mice after LPS injection. Wetested the influence of yeast RNA on the activity of NO-synthetaseferment (NOS) in blood plasma and in erythrocytes, as well as on thecontent in blood plasma of free arachidonic acid and products of itsoxide metabolism, carried out in lypoxygenase (leukotriene C4 (LTC₄))and cyclooxygenase (thromboxane B₂ (T×B₂)) ways.

Example 4.2.1 Action of Yeast RNA on the Activity of NO-Synthetase

The activity in blood plasma and erythrocytes of the enzymeNO-synthetase was measured by colometric method applied to the outcomeof reaction, nitrite anion. (Yan L., Vandivier R. W., Suffredini A. F.,Danner R. L., Human polymorphonuclear leukocytes lack detectable nitricoxide synthetase activity. J. Immunol., 15; 153(4): 1825–1834; 1994).The incubation mix (1 ml) consisted of 50 mM of HEPES (pH=7.4), 1.25 mMof CaCl₂, 1 mM of NADPH, 80 mcM FAD, 20 mcM of tetrahydrobiopterine, 13mcg/ml of calmoduline 1 mM of L-arginine, 60 mM of L-valine, 100units/ml of superoxyddismutase. HEPES is N(2hydroxyetyl)-1-piperazineethanesulfonic acid by Sigma Chemical Co.(USA), NADPH is beta-nicotinamide adenine di-nucleitide phosphate inreduced form by Sigma Chemical Co. (USA). The reaction was initiated byadding 0.1 ml of a probe containing 500 microgram of general proteindetermined by Bredford method. Incubation at 27° C. lasted for 60minutes. The reaction was terminated by adding 0.2 ml of 2N HClO₄. Themix was centrifuged at 10000 g for 10 minutes, and the supernatantliquid was used to determine the content of nitrite-anion (stablemetabolite of nitrogen oxide).

Nitrite anion was determined using the reagent of Gris in colometricreaction as decribed in Green et al. (Green L. C., Waagner D. A.,Glogowski J. et al., Analysis of nitrate, nitrite and [15N] nitrate inbiological fluids, Anal. Biochem., 126(1): 131–138;1982). The Grisreagent was prepared by mixing equal parts of 0.1% water solution ofnaphthylenediaminehydrochloride and 1% solution of sulfanilamide in 5%H₃PO₄ immediately before the measurement. The measurement was carriedout in non-protein aliquots of probe by adding Gris reagent in the 1:1proportion. In 5 minutes after mixing, the extinction at 543 hm wasmeasured. The quantity of NO₂ was measured by standard curve built forNaNO₂. The test results are presented in Table 6 below.

TABLE 6 Action of Yeast RNA and Aspirin on the Activity of NOS in MouseBlood Plasma after Carrageenan Injection (in picomol per 1 min per 1 mgof protein; M +− m; n = 5) LPS (Control) +Yeast RNA +Aspirin 0 min norm30 min 60 min 320 min 30 min 60 min 320 min 320 min M 18.41 189.45 72.03110.48 35.40 9.61 107.14 42.24 +−m 2.24 21.34 9.25 22.79 7.73 0.96 13.264.50 P1 <0.001 <0.001 <0.01 >0.05 <0.01 <0.001 <0.01 P2 <0.01<0.01 >0.5 >0.05 P1 - certainty of difference with respect to the norm(before LPS injection) P2 - certainty of difference with respect to thecontrol (without yeast RNA)

Table 6 shows that, without prior injection of yeast RNA control case, adramatic increase (more than tenfold) of NOS activity in blood plasmawas evidenced for 30 minutes after LPS was injected. Then, enzymaticactivity decreased with a later minor increase (though at a level muchhigher than normal).

A prior injection of yeast RNA in mice significantly decreased the riseof NOS activity in blood plasma during the initial stage (30 to 60minutes) of inflammatory development. This protector property of yeastRNA was not evident on the 320^(th) minute of inflammatory development,while an aspirin injection reduced NOS activity exactly during thisperiod of time.

Hence, yeast RNA has a pronounced inhibiting action on activation of theoxide way of L-arginine metabolism after the introduction of LPS, whichis expressed by inhibiting the activity of NOS in blood plasma.

Since various isoforms of NOS, both constitutive and inducible, arepresent in different nucleus cells of blood plasma: neutrophyles,thrombocytes, lymphocytes, and macrophages (Hibbs J. B., Taintor R. R.,Vavrin Z., Rachlin E. M., Nitric oxide: a cytotoxic activated macrophageeffector molecule. Biochem. Biophis. Res. Commun. 30; 157(1); 87–94;1988; Salkowski C. A., Regulation of inducible nitric oxid messengerRNA-expression and nitric oxid production by lipopolysaccharide in vivo:the role of macrophage, endogenous IFN-gamma and TNF receptor-1-mediatedsignaling. J. Immunol. 15; 158(2): 905–912; 1997) we may infer that inthe initial stage after LPS introduction (30^(th)–60^(th) minute), anactivation of the constitutive forms (neuronal and endothelial) takesplace, while the inducible form (iNOS) of blood macrophages is probablyactivated in the later period (320^(th) minute).

Further, Table 7 below shows the dynamics of changing NOS activity inmice blood erythrocytes after LPS introduction. Control animals showed aminor increase of NOS activity on the 30^(th) minute, which was replacedby a significant (almost double) decrease of NOS activity inerythrocytes.

TABLE 7 Action of Yeast RNA and Aspirin on the Activity of NOS in MouseErythrocytes after Carrageenan Injection (in picomol per 1 min per 1 mgof protein; M +− m; n = 5) LPS (Control) +Yeast RNA +Aspirin 0 min(norm) 30 min 60 min 320 min 30 min 60 min 320 min 320 min M 4.336 7.7682.323 2.232 14.245 10.213 1.146 3.613 +−m 1.105 0.999 0.383 0.515 1.1091.924 0.242 0.595 P1 >0.05 >0.1 >0.1 <0.001 <0.05 <0.05 >0.5 P2 <0.01<0.01 >0.05 <0.2 P1 - certainty of difference with respect to the norm(before carrageenan injection) P2 - certainty of difference with respectto the control (without yeast RNA)

The same dynamics of modification in NOS activity, or even a morepronounced one, was evidenced in mice erythrocytes after a priorinjection of yeast RNA. Thus, an increase of NOS activity (respectivelymore than three-fold and two-fold) was manifested on the 30^(th) and60^(th) minute. A reliable (more than three-fold) decrease of NOSactivity in erythrocytes was recorded on the 320^(th) minute of LPSaction.

Some authors (Chen L. Y., Mehta J. L., Evidence for the presence ofL-arginine-nitric oxide pathway in human red blood cells: relevance inthe effects of red blood cells on plateled function, J. Cardiovasc.Pharmacol. 32(1): 57–61; 1998) indicate that erythrocytes contain aconstitutive, Ca-dependent isoform of NOS. Thus, it is possible that theincreased activity of erythrocytal NOS during the initial stage ofinflammatory reaction, which was induced by LPS introduction, is causedby increased levels of intercellular calcium in the red cells of bloodplasma.

Example 4.2.2 Action of Yeast RNA on Oxidizing Metabolism of ArachidonicAcid

The content of free arachidonic acid (AA) was measured bytwo-dimensional thin-layer chromatography (TLC) as discussed inTsunamoto et al. (Tsunamoto K., Todo S., Imashuku S. Separation ofprostaglandines and thromboxane by two-dimensional thin-layerchromatography. J. Chromatog. 3; 417(2); 414–419; 1987. The content ofstable metabolite of thromboxane A2 (TXB₂) was studied in probes byradio-immune method with TXB₂ [³H] RIA Kit, by Amersham InternationalPLC (England) (McCann D. S., Tokarsky J., Sorkin R. P., Radioimmunoassayfor plasma thromboxane B2. Clin. Chem., 27(8): 1417–1420, 1981). Thecontent of LTC₄ was tested in probes by radio-immune method with LTC₄[³H] RIA Kit by Du Pont Ltd. Hertfordshire, (UK) (Levine L., Morgan R.A., Levis R. A. et al., Radioimmunoassay of the leukotrienes of slowreactivity substance of anaphylaxis. Proc. Natl. Acad. Sci. USA. 78(12):7692–7696; 1981).

Table 8 below demonstrates the dynamics of changes of free arachidonicacid in mice blood plasma after LPS introduction.

TABLE 8 Action of Yeast RNA and Aspirin on the Content of FreeArachidonic Acid in Mouse Blood Plasma after Carrageenan Injection (innanomol per 1 mg of protein; M ± m; n = 5) LPS (Control) +Yeast RNA 0min 30 60 320 320 +Aspirin (norm) min min min 30 min 60 min min 320 minM 2.54 2.36 3.34 3.37 1.97 1.60 2.66 2.64 ±m 0.26 0.23 0.37 0.11 0.130.16 0.20 0.13 P1 <0.1 >0.1 <0.05 >0.05 >0.02 >0.1 >0.1 P2 <0.01 <0.01<0.02 <0.01 P1 - certainty of difference with respect to the norm(before carrageenan injection) P2 - certainty of difference with respectto the control (without yeast RNA)

As shown in Table 8, the control animals demonstrated increased levelsof arachidonic acid only on the 320^(th) minute after LPS introduction.Yeast RNA evidently decreased AA content in blood plasma on the 60^(th)minute of LPS action. A decrease on the 30^(th) minute was not evident.On the 320^(th) minute after LPS introduction yeast RNA evidentlydecreased the content of AA in blood plasma in comparison with thecontrol group.

It is known that free arachidonic acid is produced when membranephospholipids are hydrolyzed with AA phospholipase, which is activatedat increased levels of free ionized calcium (Leslie C. C., Channon J.Y., Anionic phospholipids stimulate an arachinoil-hydrolyziningphospholipase A2 from macrophage and reduce the calcium requireement foractivity. Biochim. Biophys. Acta. 6; 1045(3), 261–270; 1990) Besides,there are other possible ways of releasing free AA, for example,hydrolysis of cholesterol ethers by cholesterolesterase (Moscat J.,Moreno F. Herrero C., et al., Arachidonic acid releasing systems in pigaorta endothelial cells, Biochem. Biophys Res. Commun. 30; 139(3):1098–1103; 1986). Since, the first way of synthesis of free arachidonicacid is more frequent in inflammatory processes, the test resultsindicate that yeast RNA possibly inhibits the activity of phospholipasein blood plasma.

Further, Table 9 below shows the action of yeast RNA on the contents ofthromboxane B₂, a stable metabolite of A₂ thromboxane which is producedduring oxidizing cyclooxygenase metabolism of arachidonic acid.

TABLE 9 Action of Yeast RNA and Aspirin on the Content of Thromboxane inMouse Blood Plasma after Carrageenan Injection (in picomol per 1 mg ofprotein; M +− m; n = 5) +Yeast RNA LPS (Control) 320 320 +Aspirin 0 min(norm) 30 min 60 min min 30 min 60 min min 320 min M 142.610 415.250578.775 358.240 394.940 560.813 217.602 153.903 +−m 34.210 66.600 123.8011.150 23.550 67.280 32.270 15.880 P1 <0.1 <0.2 <0.001 <0.001 <0.001<0.2 >0.5 P2 >0.5 >0.5 <0.001 <0.001 P1 - certainty of difference withrespect to the norm (before carrageenan injection) P2 - certainty ofdifference with respect to the control (without yeast RNA)

As shown in Table 9, after LPS introduction, a dramatic increase of TXB₂pools in mice plasma was evidenced on the 30^(th) and, especially, onthe 60^(th) minute. On the 320^(th) minute, the levels of TXB₂ startedto drop. Yeast RNA, like aspirin, which is a known inhibitor of thecyclooxygenase metabolism of arachidonic acid (cyclooxygenase andthromboxanesynthetase), intensifies such a decrease of TXB₂ levels aftertheir rapid increase in the early stage of inflammatory processes.

Next, Table 10 shows the dynamics of changes in the contents ofpeptidoleukotriene C4, a metabolite of lypoxygenase oxidation of AA, inmice blood plasma after LPS injection.

TABLE 10 Action of Yeast RNA and Aspirin on the Content of LeukotrieneC4 in Mouse Blood Plasma after Carrageenan Injection (in picomol per 1mg of protein M +− m; n = 5) LPS (Control) M +Yeast RNA +Aspirin +−m 0min norm 30 min 60 min 320 min 30 min 60 min 320 min 320 min P1 71.60156.64 266.33 226.78 92.18 227.00 129.35 93.99 10.72 10.03 41.09 7.4815.66 36.12 19.25 1.99 P2 <0.001 <0.01 <0.001 <0.5 <0.01 <0.5 <0.1 <0.02<0.5 <0.01 <0.001 P1 - certainty of difference with respect to the norm(before carrageenan injection) P2 - certainty of difference with respectto the control (without yeast RNA)

As shown in Table 10, the control group of animals showed an increase ofLTC₄ contents in the interval between 30^(th) and 60^(th) minute, with aslight decrease against the normal level on the 320^(th) minute. Animalstaking yeast RNA showed LTC₄ levels, which were lower than control onthe 30^(th) and 320^(th) minutes of LPS action. Aspirin showed a similarinhibiting action, which was more pronounced than the action of yeastRNA on the 320^(th) minute.

In conclusion, the test results above indicate that yeast RNA, not onlyinhibits the generation of free arachidonic acid after LPS introduction,but also inhibits its oxidation, both through lypoxygenase andcyclooxygenase.

Example 5 Anti-Inflammatory Action of Yeast RNA Based on the Model ofIschemia-Reperfusion in Rats Example 5.1 Cardioprotective Action ofYeast RNA

13 white rats with body mass 200–250 g were anesthetized with urethaneand received intra-abdominal injections at 1.25 g/kg (Kogan A. H.,Modeling the myocardial infarction, M., 1979). A tracheostome withinserted intubation pipe was placed on the rats. Artificial ventilationof lungs was provided by Vita-1 device. Skin and other tissues down tothe intercostal muscles were incised with a 2–3 mm indention from themiddle sternal line. The 4–4.5 cm. incision stretched from the jugularundercut to the sword-shaped appendix. The lower parts of the 2^(nd),3^(rd), and 4^(th) ribs, as well as the intercostal muscle between the3^(rd) and the 4^(th) ribs, were dissected by eye scissors. The initialsection of the left coronary artery is usually located in the spacebetween left auricle's eye and pulmonary cone.

A strip of myocardium sized 1.5–2 mm×1–1.5 mm was stitched up with a 3/0atraumatic needle, while going along the initial section of the artery,which could easily be seen. The revealed ligature was bandaged aroundthe artery and surrounding muscles. Then we started the observation ofthe initial macro-signs of ischemia and developing infarction. Duringthe first 10–20 seconds of ischemia, the tissue turned pale, especiallyin the upper portion of heart, and later changed partially or totally toblue (cyanosis). Contractions of the occlusion zone weakened, and itdilated. ECG's at the same standard distance from the extremities hadbeen continuously recorded during the 30 minutes of ischemia and 60minutes of reperfusion. 200 mg/kg of yeast RNA and 20 mg/kg of aspirinwere injected 30 minutes before the start of ischemia.

To determine the area and size of the post-infarction scar in rats,sections of myocardium were dyed in accordance with thep-nitrobluetetrazolium method (Mueller B., Maass B., Krause W., Witt W.,Limitation of myocardial unperfused area and necrotic zone 24 hours and7 days after coronary artery ligation in rats by the stable prostacyclinanalogue iloprost, Prostaglandins Leucot. Med. 21(3): 331–340; 1986).After reperfusion, the animals were heparinized (150 IU/kg i.v.) thehearts removed in deep ether anaesthesia and retrogradely perfused witha solution of 0.05% p-nitroblutetrazolium in phosphate buffer (30 min;100 mmHg; 37° C.). After 24 hours fixation in formaldehyde solution theventricles were weighed, transversely sectioned into 5 slices each, andan unstained area was divided from the stained myocardium and weighed.The necrotic zone was calculated.

Analysis of the necrosis zone 60 minutes after ischemia determined thatthe risk zone in the left ventricle of the heart constituted 33.3+3.4%of the left ventricle mass. In the control group, the infarction zoneconstituted 60.3+3.8% of the risk zone. Yeast RNA injection 30 minutesbefore the start of infarction on 41% decreased the proportion betweeninfarction and risk zones to 32.1%.

Analysis of ECG in ischemia-reperfussion of myocardium in rats showedthat a prior injection of yeast RNA compound decreased the amount ofextrasystols. In only one of the five rats in this group, 4 extrasystolswere detected. In the control group, which consisted of rats not treatedby yeast RNA, we registered extrasystols in 3 rats, on average 8.7+1.7.The intervals of paroxysmal tachycardia in the control group lastedlonger: 2 out of five rates had the episodes lasting for 4.2+1.3 sec onaverage.

In the group treated by yeast RNA only one rat out of five had aninterval of paroxysmal tachycardia, which lasted for 1.5 sec. ECGanalysis showed that yeast RNA improves the heart function inischemia-reperfussion of myocardium, stabilizes the leading heartsystem, and has a significant anti-arrhythmic action by decreasing thequantity of extrasystols and shortening the paroxysmal tachycardiainterval.

In conclusion, these test results show that yeast RNA has a pronouncedcardio-protector action in infarction of myocardium in rats.

Example 5.2 Action of Yeast RNA on the Activity of Myeloperoxydase inthe Ischemiazed Part of Myocardium

The mieloperoxydase activity (MPA) was studied in myocardium using themethod of Bradley et al. (Bradley P. P., Priebet D. A., Christensen R.D. et al., Measurement of cutateous inflammation: estimation ofneutrophil content with an enzyme marker, J. Invest. Dermatol., 78(3):206–209; 1982) in the modification by Grisfwold et al. (Griswold D. E.,Hillegass L. M., Hill D. E. et al., Method for quantification ofmyocardial infarction and inflammatory cell infiltration in rat cardialtissue, J. Pharmacol. Methods, 20(3): 225–235, 1988). For this purpose,the heart was extracted and rinsed in physiological solution, which wascooled to 0° C. After rinsing, a section of myocardium (1 g of thetissue) in the central zone of ischemia was cut out and frozen to −30°C. The final fraction was prepared as a 10% haemogenate with extractivebuffer containing 0.5% hexadecyltrimethyl ammonium bromide (pH 6.0) atroom temperature. Afterwards, it was centrifuged for 20 minutes at 4° C.and 12000 g.

The upper fraction (30 microliters) was used for a reaction with 0.167mg/ml of O-dianisodine in 50 millimole/l of potassium phosphate buffer(pH 6.0). The reaction was launched with adding 0.005% solution of H₂O₂.The reaction had been continuously tested for 5 minutes at 460 hm wavelength, and with readings taken every minute. A chart indicating thereadings was prepared. A unit of MPA was defined as the quantity offerment, which destroys 1 micromole/min H₂O₂ at 25° C. The data wascalculated as MPA per 1 gram of tissue.

Ischemia and reperfusion provoked an acute inflammatory response, thecentral role of which is believed to be played by neutrophils (Entman M.L., Smith C. W., Postreperfusion inflammation: a model for reaction toinjury in cardiovascular disease, Cardiovasc. Res. 28(9): 1301–1311,1994). Because the reperfusion of ischemiazed myocardium is accompaniedby intensive concentration of neutrophils within the risk zone (HearseD. J., Bolli R., Reperfusion induced injury: manifestation, mechanismsand clinical relevance. Cardiovasc. Res. 26(2): 101–108; 1992), Whichreleases various inflammatory mediators, such as free oxygen radicals,cytokines, and haemokines, and increases ischemic-perfusion damages ofmyocardium (Entman M. L., Michael L., Rossen R. D., et al. Inflammationin the course of early myocardial ischemia, FASEB J., 5(11): 2529–2537;1991), and a direct link exists between the intensity of concentrationof neutrophils in ischemiazed myocardium and the activity ofmieloperoxydase, a special ferment contained in neutrophils, so that anincrease of MPA activity directly correlates with the quantity ofleukocytes migrating to the inflammation zone.

Analysis of the activity of myeloperoxydase in the ischemized sector ofmyocardium after 30 minutes of occlusion and 1 hour of reperfusion ofthe left coronary artery in rats, showed that it is equal to 211.8+16.7units per 1 g of tissue in the control group. When the animals wereinjected with aspirin, the activity decreased to 176.1+5.9. RNA-Dinjection decreased the activity by one third to 152.3+9.8 units per 1 gof tissue.

Further, an intravenous dose of 200 microgram/kg of yeast RNA in rats,injected 30 minutes before ischemia, decreased the concentration ofneutrophils in the risk zone after an hour-long reperfusion. Thequantity of neutrophils decreased approximately by 30%, which is twicethe result obtained for aspirin (20 microgram/kg). This allows us toconclude that the yeast RNA will be effective when used as acardio-protector in cases of ischemia and myocardial reperfusion.

Example 5.3 Action of Yeast RNA on the Activity of NOS in Ischemia Cases

The tests were conducted on rats with an infarct of myocardiumexperimentally induced by occlusion of the coronary artery for 30minutes. Blood was taken from the coronary artery and from the heartwhich was divided into the intact zone, border zone, and infarctionzone. The activity of NOS ferment was measured in different heart zonesand in blood. Also, we measured the contents of free arachidonic acid(heart and blood) and products of its oxidizing metabolism blood). Thetest results are shown in Table 11 below.

TABLE 11 Action of Yeast RNA on the Activity of NOS in Rat Heart inIschemia (in picomol per 1 mg of protein; M +− m; n = 5) Ischemia 30 min(Control) Ischemia 30 min + Yeast RNA Infarction Border Infarction NormBorder zone zone Intact zone zone Together M 46.500 259.310 185.626129.655 59.634 115.122 122.630 +−m 7.000 60.683 48.635 30.341 11.64940.509 26.413 P1 <0.01 <0.05 <0.2 <0.5 <0.1 >0.05 P2 <0.2 <0.5 <0.05P1 - certainty of difference with respect to the norm (before ischemia)P2 - certainty of difference with respect to the control (without yeastRNA)

The data in Table 11 demonstrates that, during a short-term ischemia,the activity of NOS increased more than three-fold in the infarctionzone (115±40 and 186±49 pmol/min on 1 mg of protein accordingly, in thetest and control groups). Hence, yeast RNA almost completely normalizedthe activity of NOS in the border zone of ischemic heart infarction,which may be one of the mechanisms of its cardio-protecting action.

Since cardiomyocytes contain both the inducible NOS isoform and itsconstituent isoforms (Balligand J. L., Kobzik L., Han X., et al., Nitricoxide-dependent parasympathetic signaling is due to activation ofconstitutive endotelial (type III) nitric oxid synthetase in cardiacmyocytes, J. Biol. Chem., 16; 270(24); 14582–14586; 1995; Peng H. B.,Spiecker M., Liao J. K. Inducible nitric oxid: an autoregulatoryfeedback inhibitor of vascular inflammation, J. Immunol. 15; 161(4):1970–1976; 1998; Oddis C. V., Simmons R. L., Haffler B. G., Finkel M. S.cAMP enchances inducible nitric oxid synthase mRNA stability in cardiacmyocytes, Am. J. Physiol. 269(6): H2044–2050; 1995), taking into accountthe short period (30 minutes) of ischemia, it is inferred that yeast RNAinhibits the constituent isoform of NOS. At the same time, since iNOS ispresent, yeast RNA may also act as an inhibitor of activity of inducibleNOS in ischemic cardiomyocytes.

Further, the influence of yeast RNA on NOS activity in rat blood inischemia is shown in Table 12 below in Example 5.4. The test results inTable 12 show that, unlike in the heart, the activity of NOS in theblood of control animals in ischemia decreased twice (14.22+1.43 and30.35+3.40 pmol per 1 mg of protein accordingly in ischemia andnormoxia), which is usual for hypoxia (Arnet W. A., McMillan A.,Dinerman J. L. et al., Regulation of endothelial nitric oxid synthaseduring hypoxia, J. Biol. Chem. 271(25): 15069–15073; 1996). Introductionof yeast RNA almost completely normalized NOS activity in rat bloodafter a 30-minute ischemia.

Example 5.4 Action of Yeast RNA on the Oxidizing Metabolism ofArachidonic Acid in Ischemia Cases

Table 12 below shows the content of NOS and free arachidonic acid in theblood of normal and ischemic animals.

TABLE 12 Action of Yeast RNA on the Activity of NOS and Content ofArachidonic Acid in Rat Blood in Ischemia NOS Activity in picomol per 1mg protein; M +− m; Content of free Arachidonic acid n = 5 (nmol/1 mg ofprotein) Ischemia + Ischemia Ischemia Yeast 30 min Ischemia + Norm 30min RNA Norm (control) Yeast RNA M 30.35 14.22 26.45 0.77 0.24 0.48 +−m3.40 1.43 3.73 0.13 0.04 0.02 P1 <0.01 <0.5 <0.01 >0.05 P2 <0.05 <0.01P1 - certainty of difference with respect to the norm (before ischemia)P2 - certainty of difference with respect to the control (without yeastRNA)

As shown in Table 12, the control group of animals demonstrateddecreased levels of free AA more than three-fold (0.77±1.43 and30.35±3.40 nmol/min on 1 mg of protein accordingly in normoxia andischemia cases). The introduction of yeast RNA somewhat normalized thecontent of AA, by increasing it twice against the control value(P<0.001).

Table 13 indicates the content of free arachidonic acid in differentheart zones in ischemia cases.

TABLE 13 Action of Yeast RNA on the Content of Free Arachidonic Acid inRat Heart in Ischemia (in nmol per 1 mg of protein; M − m; n = 5)Ischemia 30 min (Control) Ischemia 30 min + Yeast RNA Infarction BorderInfarction Norm Border zone zone Intact zone zone Together M 4.827 9.9109.716 9.813 7.270 8.530 7.900 +−m 0.378 1.003 0.947 0.919 0.456 0.7410.493 P1 <0.01 <0.01 <0.001 <0.01 <0.01 <0.01 P2 >0.05 >0.05 >0.5 P1 -certainty of difference with respect to the norm (before ischemia) P2 -certainty of difference with respect to the control (without yeast RNA)

As shown in Table 13, the control group of animals demonstrated areliable (more than two-fold, P<0.01) increase of AA levels both in theborder and infarction zones of rat hearts. The introduction of yeast RNAsomewhat decreased the level of arachidonic acid in both heart zones,but the difference was not evident (P>0.05).

Table 14 below shows the action of yeast RNA on the levels ofeicosanoids in rat blood in ischemia cases.

TABLE 14 Action of Yeast RNA Compound on Eukosanoids in Rat Blood inIschemia (in picomol per 1 mg of protein; M − m; n = 5) Thromboxane B2Leucotriens C4 Ischemia Ischemia + Ischemia 30 min Yeast 30 minIschemia + Norm (control) RNA Norm (control) Yeast RNA M 53.00 130.7262.49 24.10 39.62 25.69 +−m 10.67 33.92 7.98 3.94 10.18 4.04 P1<0.05 >0.5 <0.2 >0.5 P2 <0.1 <0.5 P1 - certainty of difference withrespect to the norm (before ischemia) P2 - certainty of difference withrespect to the control (without yeast RNA)

As shown in Table 14, the control group of animals demonstrated moreincreased levels of products of cyclooxygenase reaction of TBX₂ (morethan two-fold, but the difference is not evident—P>0.05) rather than thelevels of the product of lipoxygenase reaction LTC₄ (P<0.2). Theintroduction of yeast RNA almost completely normalized that content ofeicosanoids in rat blood in ischemia cases.

In conclusion, in addition to the modulating influence on NOS activityin ischemia (inhibition in cardiomyocytes and, on the contrary, increasein blood), the cardio-protecting action of yeast RNA may also bemediated by modulating the oxidizing metabolism of arachidonic acid.

Example 6 Anti-Inflammatory Action of Yeast RNA Based on the Model ofAuto-Immune Pathology Model (Adjuvant Arthritis) In Vivo

Adjuvant arthritis develops after rats are injected with Freud'sadjuvant and is a part of the generalized process, which is accompaniedby the impairment of bone and connecting tissues. Morphological testsshow, that during the development of adjuvant arthritis,inflammatory-degenerative changes emerge in tissues surrounding thejoint as well as inside the articular bursa and in joint cartilages. Itis believed that this inflammatory reaction has all the properties of animmunologic process and constitutes a delayed immune reaction to amicrobe antigen. The pathological process of adjuvant arthritis is verysimilar to arthritis in humans.

Example 6.1 Action of Yeast RNA in an Auto-Immune Pathology Model(Adjuvant Arthritis)

Adjuvant arthritis was modeled in rat males according to Courtright etal. (Courtright L. J., Kuzell W. C., Sparing effect of neurologicaldeficit and trauma on the course of adjuvant arthritis in the rat, Ann.Reum. Dis. 24(4): 360–368; 1965). Control animals received a singlehypodermic dose of 0.1 ml of standard Freud's adjuvant in the distalpart of the tail. Adjuvant arthritis developed on the 14–20^(th) dayafter the injection. Arthritis symptoms were determined by X-rays: adarkened area and shadows around the joints of back legs imply astarting impairment of the joint and gristle tissue.

In the test group yeast RNA was diluted in a 0.9% concentration of NaCl,was injected intra-abdominally in the concentration 100 mg per rat, aday before the injection of Freud's adjuvant. Yeast RNA has been alsointroduced after adjuvant's injection in three series within 4 days withthree-day intervals.

Results of the analysis showed that arthritis in the control groupstarted to develop on the 14^(th) day and was manifested byexudative-proliferate growth of the synovial capsule and gristleimpairment. On the 20^(th) day, a hardening of tissue around the jointwas witnessed and fibrosis of the synovial capsule started. On the30^(th) day, the ruining of gristle becomes evident. In the test group,which took yeast RNA, no signs of arthritis were witnessed for 20 days.Arthritis symptoms, similar to the ones witnessed in the control groupon the 14^(th) day, appeared only on the 30^(th) day.

During the development of adjuvant arthritis, back legs became larger inthe control animals. In particular, on the 30^(th) day in the controlgroup, the size of back legs evidently increased by 1.04 millimeter(4.9±0.13 in comparison with 3.86±0.1 at the beginning of experiment).In the test group, legs grew only by 0.24 millimeter (4.1±0.11 on the30^(th) day of experiment from 3.96±0.08 at the beginning ofexperiment). Hence, yeast RNA delays the development of adjuvantarthritis, which is also supported by a decreased growth rate of backlegs.

Example 6.2 Action of Yeast RNA on the Activity of NOS in Rat Blood inan Auto-Immune Pathology (Adjuvant Arthritis)

The activity of NOS was evaluated in the blood of normal rats and, onthe 3^(rd)(I), 8^(th)(II), and 14^(th)(II) day in the course of anauto-immune pathology in the control group of rats (i.e., not takingyeast RNA) and in the test animals, which were injected with yeast RNA.The results are shown in Table 15 below.

TABLE 15 Action of Yeast RNA Compound on the Activity of NOS in RatBlood in the Dynamics of Adjuvant Arthritis (in picomol per 1 min per 1mg of protein; M ± m; n = 5) Control +Yeast RNA I II III I II III Norm3^(rd) 8^(rd) 14^(rd) 3^(rd) 8^(rd) 14^(rd) M 30.650 236.760 24.340111.540 70.000 40.660 33.960 +−m 7.352 76.418 8.596 15.777 9.245 5.0526.036 P1 <0.05 >0.5 <0.01 <0.02 <0.5 >0.5 P2 <0.02 <0.1 <0.01 P1 -certainty of difference with respect to the norm (in adjuvant arthritis)P2 - certainty of difference with respect to the control (without yeastRNA)

As shown in Table 15, the control group of animals showed a substantialincrease of NOS activity on the 3^(rd) and 14^(th) day of auto-immunepathology in comparison with norm (30.65±7.35 picomol per 1 mm per 1 mgof protein in norm, 236.76±76.42 picomol per 1 min per 1 mg of proteinon the 3^(rd) day, and 111.54±15.78 picomol per 1 mm per 1 mg of proteinon the 14^(th) day). Such a significant increase in the activity of NOSindicates that activity of the inducible NOS-isoform (iNOS), whosesynthesis is initiated by anti-inflammatory cytokines INE-γ, IL-1β,TNF-α, et. al., is the main compound in the calculated activity of NOS.

In the period between the 3^(rd) (initiation of the auto-immune process)and 14^(th) day (development of pathology), we observed a normalizationin the activity of NOS in blood (24.34±8.60 pmol per 1 mm per 1 mg ofprotein). This may probably be attributed to the activated protectivereaction of body, and could be induced by inhibition of the expressionof NOS as well as by modulation of the stability of its mRNA, or byinhibiting the process of its translation.

In the group of animals which took yeast RNA, initiation of theauto-immune process (on the 3^(rd) day) was accompanied by a muchsmaller (in comparison with the control group) increase in the activityof NOS in blood (70.00±9.24 pmol per 1 mm per 1 mg of protein against236.76±76.42 pmol per 1 mm per 1 mg of protein). Moreover, the activityof NOS decreased progressively over the next period in development ofauto-immune process (40.66±5.05 pmol per 1 mm per 1 mg of protein on the8^(th) day and 33.96±6.04 pmol per 1 mm per 1 mg of protein on the14^(th) day).

Therefore, our tests on changes in the activity of NOS in rat blood inthe course of an auto-immune process lead to the conclusion that yeastRNA is effective in decreasing the activity of iNOS in the course of anauto-immune process, both during its initiation and in the chronicstage. This property allows the use of yeast RNA in pathologicalconditions which are accompanied by iNOS induction: inflammatoryprocesses, diabetes, atherosclerosis, tumour, hepatitis, infections,neuro-degenerate diseases (Parkinson's disease, Alzheimer's disease,multiple sclerosis, encephalitis), and others.

Example 6.3 Membrane-protecting Action of Yeast RNA

The tests were conducted in vivo on the model of a chronic auto-immuneprocess, which was accompanied by generation of a great quantity of freeradicals (especially, nitric oxide) during the early stage ofinitiation. The membrane-protecting action of yeast RNA was studied byevaluating acid resistance of erythrocytes in the course of anauto-immune process. Acid resistance characterizes the wholeness oferythrocytal membranes. It increases in the chronic stage of differentpathologies and decreases in the acute stage of development (process ofinitiation). For example, in the early period of development ininflammations, free-radical processes, which are induced by a generationof free radicals of oxygen and nitrogen, including nitric oxidegenerated by the inducible isoform of NOS (iNOS), are highly activated.

The level of damage in erythrocytes under the influence of variousharmful factors in the course of an auto-immune process was evaluated bykinetic indicators of hemolysis, induced by a pH decrease in theenvironment. Kinetic indicators of hemolysis were recorded; the numberof damaged cells was determined spectrophotometrically in equal periodsof time (30 s) by changes in the value of integral light dispersion oferythrocytal suspension (λ=750 nmol). Absorption spectra were registeredby a spectrometer SF-26 (Russia). Acid lysis of erythrocytes wasinitiated by adding 10 μl of blood, which was diluted 20 times in theisotonic medium 0.14 mol of NaCl+0.01 mol of the citrate-phosphatebuffer with pH=2.0–3.5 (volume: 1 ml; density of erythrocytes insuspension: 0.7×10⁶ cells per ml). For such densities, the value ofintegral light dispersion of erythrocytes depends on the count, size,and shape of cells and is proportional to the number of cells insuspension.

Results are represented in the diagram of acid hemolysis of erythrocytesin Table 16 below, as the integral parameter of this process: totalnumber of acid resistance of erythrocytes was calculated by summing upthe products of the number of cells a_(I) which hemolyzed over theperiod of time a_(j) and t_(j) (total resistance (I)=Σa_(i)·t_(i)).

Decreased extinction levels on hemolysis diagrams represent thesuccession of erythrocytes with increased resistances enteringhemolysis. Extinction starts decreasing usually 1.5–2 min later after ahemolytic injection (1 ml 0.004N HCl, which was prepared from 0.1N HCland checked by titration). A lag-period of hemolysis is caused by apre-hemolysis change in the form of erythrocytes (spherulation).Hemolysis of a single erythrocyte does not exceed 10 seconds. Hence, a30-second interval between the measurements of existence levels excludesthe possibility of counting twice the same erythrocyte undergoing lysis.It follows that, by the photometric registration of hemolysis kinetics,we can calculate, from the derived series of existences with intervals30 seconds, the percentage of distribution of erythrocytes by resistancegroups.

The change of existence from the beginning of hemolysis (E_(b), t_(b))to its final completion (E_(e), t_(e)) is proportional to the number ofall cells involved in hemolysis (100%), hence:ΔE=E _(e) −E _(b)=100%.

This total quantity of erythrocytes which undergo hemolysis (100%)consists of the quantity of erythrocytes which undergo hemolysis each 30seconds (E_(i+1)−E_(i)) in the interval t_(e)−t_(b)=duration ofhemolysis:ΔE=ΔE _(i+1) −E _(i)=100%.

The results are shown in Table 16 below.

TABLE 16 Action of Yeast RNA Compound on the Acid Resistance ofErythrocytes in the Dynamics of Adjuvant Arthritis (Total Resistance; M+− m n = 5) Control +Yeast RNA I II III I II III Norm 3^(rd) 8^(rd)14^(rd) 3^(rd) 8^(rd) 14^(rd) M 712.333 95.400 448.600 1013.800 372.600638.800 565.800 +m 85.429 37.776 95.843 290.509 72.667 78.903 80.244 P1<0.01 <0.1 <0.5 <0.05 >0.5 >0.5 P2 <0.02 <0.2 <0.2 P1 - certainty ofdifference with respect to the norm (in adjuvant arthritis) P2 -certainty of difference with respect to the control (without yeast RNA)

As shown in Table 16, there is total resistance of non-showered raterythrocytes in the course of an auto-immune process. This indicator isequal to 712±85 for normal erythrocytes. During the initiation of anauto-immune process, total resistance of erythrocytes decreased 7 timesand constituted 95±38 units. It was gradually increasing in the courseof pathology and reached 1114±290 units on the 14^(th) (III) day.

Such a significant decrease in the acid resistance of erythrocytesindicates substantial changes in plasmatic cellular membranes, which isperhaps due to the oxidation of protein and lipid membrane components byfree radicals, including nitric oxide, which are actively generated inthis period, and in plasma, from which we can infer a modulation in thecontents of free cholesterol, polyamines, and other stabilizers, as wellas increased levels of destabilizers, such as polyunsaturated free fattyacids.

Animals which took yeast RNA during the initiation of an auto-immuneprocess did not have such decreased acid resistance of erythrocytes asin the control group. Total resistance was equal to 373±73 units, which,though lower than the norm (P<0.05), is greater than in the controlgroup (P<0.05). During the later periods in development of auto-immunepathology, total resistance of erythrocytes in animals taking yeast RNAwas at the normal level.

Therefore, yeast RNA has immune-stabilizing action. Taking into accountthe main mechanisms of damage in this pathology, which are oxide stressand damage of plasmatic membrane components by free-radicals, we canalso conclude that yeast RNA is anti-radical.

Example 7 Action of Yeast RNA on Blood Indicators

Blood samples, taken from patients before and after the treatment withyeast RNA compound, were studied on the automatic hemocytometer “Seronol1900”, Austria in accordance with producer's recommendations. First, thequantities of leukocytes [WBC], erythrocytes [RBC], and thrombocytes[PLT] in 1 microliter of blood, quantity of hemoglobin [HGB] in g/l,neutrophils (NTP) and hematocrite [HCT] in percentage, were measured.Patients and healthy volunteers were selected after a preliminaryanalysis of the mentioned above indicators. Groups of 4 to 6individuals, who showed a strong reduction of one or more indicatorsmentioned above in comparison with normal levels, were selected forfurther analysis. Depending on the type of condition, treatment wasconducted from 1 to 18 weeks. Yeast RNA compound was administered eitherin capsules, in the concentration of 250 mg of yeast RNA per capsule, orin suppositories, in the concentration of 1.0 g of yeast RNA persuppository.

Example 7.1 Influence of Yeast RNA on Blood Cytopenia in RelativelyHealthy Individuals and Athletes

The treatment lasted between 10 days and 6 weeks. The compound wasadministered in capsules, 1 capsule per day, or in suppositories, 1suppository per three days. Tests were conducted 1–2 times a week, andthe results are represented in Tables 17 and 18. Table 17 shows theresults of treatment of a group of patients with the symptoms of anemia,who took the compound of yeast RNA in capsules 1 g/day for 3 weeks. Itis apparent from the tables that treatment resulted in a stable increaseof hemoglobin concentration from 12.0 g/l to 14.0 g/l, and hematocritepercentage from 30% to 37%. The same group also showed an increase inthe concentration of RBC by 17.7% along with the increase in quantitiesof WBS and PLT, accordingly by 26.5% and 59.9%.

TABLE 17 Influence of Yeast RNA (in capsules 1 g/day) on Blood Cytopeniain Relatively Healthy Individuals. 4 days 11 days 21 days Before of ofof Blood treat- treat- treat- treat- tests Norm ment ment ment ment WBC4.3–10.8 thous./mkl 6.28 5.78 7.20 7.95 M± 0.512 0.512 0.531 1.090 M RBCM± <4.0 mln/mkl 3.77 4.13 4.34 4.44 M 0.117 0.137 0.124 0.188 PLT M±200–300 thous./mkl 211.75 224.00 266.00 338.75 M 13.931 12.623 9.22944.205 HGB M± 14–16 g/l 12.95 13.78 14.55 13.90 M 0.433 0.585 0.5560.438 HCT M± 43–47% 30.00 31.58 34.68 37.35 M 0.599 1.009 0.986 1.767

Since the usage of compound per os leads to its fast hydrolysis, muchsmaller concentration actually gets into the blood. Therefore, theinfluence of yeast RNA compound in suppositories in the concentration 1g per day was studied in the group of relatively health individuals.Suppositories were taken on the 1^(st), 3^(rd), and 6^(th) days. Theresults of studies show that hematocrite increased from 33.2% to 41.5%,while hemoglobin increased from 13.5 g/l to 14.5 g/l. The quantity ofleukocytes increased by 46.5%, and erythrocytes by 10.6%. The quantityof thrombocytes remained stable. Therefore, the usage of yeast RNAcompound in the form of suppository, which is equated to the intravenousinjection of the compound, allows to achieve the same results threetimes as fast as with capsules (7 days with suppositories versus 21 daywith capsules) and with a sevenfold reduction in the total concentrationof yeast RNA for the duration of treatment from 21 g to 3 g. Suchaccelerated normalization of blood indicators is important duringbleeding, when blood transfusion is required.

Table 18 shows the results of treatment of decreased blood indicators inathletes. It is known, that during intense practice, such individualsoften have decreased blood indicators: hematocrite, hemoglobin, andothers. The compound of yeast RNA was administered at 1.5 g per day for25 days. Hematocrite and hemoglobin levels were measured.

TABLE 18 Influence of Yeast RNA (in capsules 1.5 g/day) on BloodQuotients in Athlets 3 days before 10 days of 18 days of 25 days ofNormal treatment Day of treatment treatment treatment treatment HGB14–16 g/l 13.88 13.55 14.13 14.30 14.85 M± 0.678 0.685 0.611 0.688 0.584m HCT 43–47% 42.80 40.60 43.78 45.70 46.57 M± 1.672 1.272 1.664 1.8571.707 m

Thus, it was demonstrated that hematocrite and hemoglobin levelsdecreased during the 2 days of intense practice from 42.7% and 13.9 g/lto 41.0% and 13.5 g/l accordingly. 10 days after the use of yeast RNA,they increased to 43.7% and 14.1 g/dl; after 16 days—to 45.7% and 14.3g/dl accordingly, and after 21 day—to 46.5% and 14.8 g/dl. Therefore,this group showed after 3 weeks of intense training increase ofhematocrite by 13.4%, and hemoglobin—by 6%.

Example 7.2 Influence of Yeast RNA on Blood Cytopenia in Cancer Patients

Anemia plays a very negative role in cancer patients, especially afterchemotherapy or radiotherapy. Patients with hemoglobin levels below 8g/l are often not allowed chemotherapy or radiotherapy at all. A seriesof researches have shown that treatment of anemia and increase ofhemoglobin levels are crucial in the treatment of cancer patients, whichundergone chemotherapy (J. W. Adamson, H. Ludwig. Predicting theHematopoietic Response to Recombinant Human Erythropoietin (EpoetinAlfa) in the Treatment of the Anemia of Cancer, Oncology, Vol. 56, pp.46–53 (1999)).

By using erythropoietin in the treatment of patients, who undergonechemotherapy, it has been shown that the increase of hemoglobin from 8g/dl to 10 g/dl is accompanied by a modest improvement in quality oflife measures in cancer patients. A more significant improvement in thequality of life measures occurs after the increase of hemoglobin from 10g/l to 12 g/l. Hence, hemoglobin level of 12 g/l is optimal for cancerpatients, both in accordance with their quality of life measures andtreatment results (J. Crawford, Anemia, Fatigue, and Erythropoietin,42^(nd) Annual Meeting of the American Society of Hematology, 2000,Medscape, Inc.).

A group of cancer patients was studied, in which the patients took 1–2 gof yeast RNA compound per day in capsules for 8 days beforechemotherapy, during chemotherapy, in the break between, and afterrepetitive chemotherapy. Blood tests were conducted in accordance withthe method described above. Results of the analysis are presented inTable 19.

TABLE 19 Influence of Yeast RNA (in capsules 1–2 g/day) on BloodCytopenia in Cancer Patients. Blood Before 1 Week 2 Weeks 4 Weeks 5Weeks 6 Weeks tests Norm treatment treatment treatment treatmenttreatment treatment WBC 4.3–10.8 5.73 5.20 4.53 5.10 5.10 4.20 M±thous./mkl 2.448 1.079 0.180 0.715 1.373 0.091 M RBC <4.0 mln/mkl 2.583.10 3.38 3.43 3.85 3.90 M± 0.131 0.147 0.131 0.180 0.218 0.204 M PLT200–300 84.00 93.75 125.00 166.25 161.25 193.75 M± thous./mkl 6.06918.414 20.207 19.512 7.181 5.543 M HGB 14–16 g/l 67.25 85.75 92.75 98.75110.75 121.00 M± 4.423 3.945 4.768 6.369 6.356 1.958 M HCT 43–47% 22.7531.00 33.00 36.00 36.75 40.00 M± 1.109 3.109 1.472 2.739 2.626 0.707 M

As reported in Table 19, from the start of treatment with yeast RNAcompound, the patients show a stable increase in hemoglobin levels,hematocrite percentage, quantities of erythrocytes and thrombocytes.During the 6 weeks of treatment, the quantity of hemoglobin increasedalmost twice, from 67 g/l to 121 g/l, the quantity of erythrocytesincreased by 51%, and thrombocytes by 130%. The patients did not show adeterioration in other blood indicators, and reported an increased senseof well-being with the increase of hemoglobin levels to 120 g/l.

Example 7.3 Influence of Yeast RNA on Blood Cytopenia in HIV-infectedPatients

HIV-infected patients have a complex malfunction of hematopoiesis,leading to a de-normalization of all 3 cell lines which originate fromthe hematopoietic progenitor cell. Therefore, at least 80% ofHIV-infected patients become anemic in the process of development of theHIV-infection, more than 50% have neutropenia, and about 40% hasthrombocytopenia. Hence, cytopenia of HIV-infected individuals is one ofthe first signs of the infection (A. M. Levin, Anemia, Neutropenia, andThrombocytopenia: Pathogenesis and Evolving Treatment Option inHIV-Infected Patients, HIV Clinical Management, Vol. 10, 1999, Medscape,Inc.).

A group of selected HIV-infected patients was studied under medicalsupervision for about 6 months. After analysis of their hematopoiesis,patients started treatment with yeast RNA capsules in the concentrationof 1 to 2 g per day for 18 weeks. Hematological and biochemical testswere conducted every 1–2 weeks. Results of treatment are represented inTable 20. It was found that HIV-infected patients in the past 6 monthshad a pronounced cytopenia with decreased levels of hemoglobin,quantities of thrombocytes, neutrophiles, and the percentage ofhematocrite. A number of patients had hepatitis. Between the 12^(th) and14^(th) weeks of treatment with yeast RNA, because of an epidemic, thepatients got influenza, which lead to the inflammation of lungs.Therefore, additional treatment by antibiotics was used during thisperiod.

TABLE 20 Influence of Yeast RNA (in capsules 1–2 g/day) on BloodCytopenia in HIV-Infected Patients Be- fore Bl. treat- Weeks aftertreatment tests Norm ment 2 4 6 8 10 12 14 16 18 WBC 4.3–10.8 3.28 ±4.00 ± 5.10 ± 4.35 ± 5.2 ± 5.9 ± 5.25 ± 5.38 ± 5.35 ± 5.15 ± M ± mth./mkl 0.1 0.3 0.1 0.3 0.07 0.1 0.2 0.3 0.08 0.08 RBC <4.0 3.48 ± 3.83± 4.00 ± 4.20 ± 4.1 ± 4.3 ± 4.15 ± 4.30 ± 4.33 ± 4.33 ± M ± m mln/mkl0.2 0.1 0.4 0.2 0.2 0.2 0.2 0.2 0.2 0.1 PLT 200–300 121.2 ± 132.5 ±192.5 ± 185.0 ± 247.5 ± 196.5 ± 208.7 ± 216.2 ± 207.5 ± 223.7 ± M ± mth./mkl 9.2 7.5 29.8 11.9 26.2 22.3 4.2 8.9 8.5 32.8 NTP 34–83% 36.75 ±44.00 ± 49.75 ± 34.5 ± 46.7 ± 54.0 ± 46.5 ± 46.0 ± 53.0 ± 43.0 ± M ± m3.1 2.3 3.1 0.6 3.0 3.7 2.9 3.1 0.9 4.7 HGB 14–16 99.43 ± 109.6 ± 125.0± 130.9 ± 129.8 ± 133.2 ± 127.5 ± 128.9 ± 132.8 ± 125.6 ± M ± m g/l 4.24.4 6.1 8.1 8.4 9.1 5.5 5.5 6.7 3.1 HTC 43– 30.5 ± 37.75 ± 35.5 ± 42.5 ±44.2 ± 43.0 ± 43.7 ± 41.5 ± 42.7 ± 40.7 ± M ± m 47% 1.0 1.4 4.6 0.8 0.61.47 3.2 3.0 3.1 1.1

As indicated in Table 20, treatment resulted in a stable normalizationof all blood indicators in HIV patients in 4–6 weeks. Normalization oferythrocytes, neutrophiles, and thrombocytes became visible in 4 weeks,corresponding to a complex normalization of the differentiation of all 3cell lines of progenitor CFU-GEMM. No abnormal stimulation of thequantity of lymphocytes was detected. Hemoglobin increased in 4 weeksfrom 99.43 g/l to 125 g/l and remained stable until the end of tests.Neutrophiles increased in 4 weeks from 36.75% to 49% and remained withinthese limits with certain deviation in the next 18 weeks.

Thus, treatment of HIV-infected patients with yeast RNA resulted in astable normalization of cytopenia. This allows to conclude that thecompound can be successfully used for prevention and treatment ofinflammatory processes and to maintain the capacity for work and qualityof life measures in patients at a high level.

1. A method of protecting erythrocytes, which comprises administering toa mammal in need of such treatment an effective amount of ribonucleicacid extracted from yeast and an acceptable vehicle, carrier, ordiluent, wherein said ribonucleic acid extract has been purified so thatit comprises more than 14.5% by weight nitrogen and more than 8.5% byweight phosphorus, and wherein the ribonucleic acid extract isadministered to a relatively healthy individual or athlete showingsymptoms of anemia, a cancer patient, or an HIV-infected patient.
 2. Amethod in accordance with claim 1, wherein said ribonucleic acid isadministered in an amount within a range of from 0.1 mg to 1 g per kgweight of a mammal.
 3. A method in accordance with claim 1,characterized in that said ribonucleic acid is administered in an amountwithin a range of from 0.1 to 1 g.
 4. A method in accordance with claim1, wherein said ribonucleic acid is obtained from a Saccharomycescerevisiae.
 5. A method in accordance with claim 1, wherein saidribonucleic acid is obtained from a Candida utilis.
 6. A method inaccordance with claim 1, wherein said ribonucleic acid is administeredby an intradermal, hypodermal, oral, intra-abdominal, intramuscular, orintravenous route.
 7. A method in accordance with claim 1, wherein saidribonucleic acid is administered in the form of capsules.
 8. A method inaccordance with claim 1, wherein said ribonucleic acid is administeredin the form of suppositories.
 9. A method in accordance with claim 1,wherein the ribonucleic acid extract is administered to a cancer patientor an HIV-infected patient showing symptoms of blood cytopenia, and theblood cytopenia is selected from the group consisting of anemia,thrombocytopenia, neutropenia, and combinations thereof.
 10. A method inaccordance with claim 9, wherein the blood cytopenia is anemia.
 11. Amethod in accordance with claim 9, wherein the blood cytopenia isthrombocytopenia.
 12. A method in accordance with claim 9, wherein theblood cytopenia is neutropenia.